NADPH-Oxidase in Active Multiple Sclerosis Lesions · 1.1 Multiple Sclerosis and its pathology Multiple Sclerosis (MS) is the most common neurological disease in young adults in the

NADPH-Oxidase in Active Multiple Sclerosis Lesions

Doctoral thesis at the Medical University of Vienna for obtaining the academic degree

Doctor of Philosophy

Submitted by

Maga Marie-Therese Fischer

Supervisor: Prof. Hans Lassmann

Center for Brain Research Division of Neuroimmunology Spitalgasse 4,Vienna, A-1090

Vienna, 11/2011

i

Tabel of contents

Abstract…………………………………………………………………………………………………………………..…………..iii

Zusammenfassung……………………………………………………………………………………………………………….iv

1 Introduction ....................................................................................................................1

1.1 Multiple Sclerosis and its pathology .........................................................................1

1.2 The Architecture of Inflammatory Demyelinating Lesions.........................................3

1.2.1 Structural Architecture of Classical Active Lesions: ............................................4

1.2.2 Structural architecture of slowly expanding lesions (SEL): .................................7

1.3 Inflammation as a source for reactive oxygen intermediates ....................................9

1.3.1 Reactive oxygen species (ROS) formation and detoxification .............................9

1.3.2 Activated microglia and macrophages as sources of ROS .................................10

1.3.3 Oxidative phosphorylation as a source of ROS .................................................11

1.3.4 Mitochondrial injury and neurodegeneration ..................................................11

1.3.5 Oxidative damage in Multiple Sclerosis lesions ................................................13

2 Material and Methods ..................................................................................................15

2.1 Human autopsy tissues ...........................................................................................15

2.2 Immunohistochemistry ..........................................................................................17

2.2.1 IHC with ABC-System .......................................................................................17

2.2.2 IHC for Oxidized Phospholipids ........................................................................18

2.2.3 Klüver-Barrera- PAS .........................................................................................19

2.2.4 Markers used ..................................................................................................19

2.2.5 Double staining for light microscopy ...............................................................20

2.2.6 Double staining for fluorescence microscopy ..................................................21

2.2.7 Quantitative analysis .......................................................................................23

2.2.8 Statistical analysis............................................................................................23

2.3 Whole genome arrays ............................................................................................24

2.3.1 Assessment of RNA quality: In situ hybridization .............................................24

2.3.2 Scratching of areas of interest .........................................................................27

2.3.3 RNA Isolation and Amplification ......................................................................27

2.3.4 Agilent arrays ..................................................................................................29

2.4 RNA quality control ................................................................................................30

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2.5 Isolation, culturing and staining of Astrocytes ........................................................32

2.5.1 Astrocyte-enriched cultures ............................................................................32

2.5.2 Treatment of astrocyte-enriched cultures .......................................................34

2.5.3 Immunocytochemical staining and analysis .....................................................34

3 Results ..........................................................................................................................35

3.1 The project .............................................................................................................35

3.2 NADPH Oxidase Expression in Active Multiple Sclerosis Lesions in Relation to

Oxidative Tissue Damage and Mitochondrial Injury ...........................................................36

3.2.1 The importance of lesional staging ..................................................................37

3.2.2 Microarray studies ..........................................................................................38

3.2.3 Difference in gene expression between different cases in relation to lesion

activity ............................................................................................................39

3.2.4 Mitochondrial genes........................................................................................40

3.2.5 Genes involved in radical production and response to oxidative stress ...........43

3.2.6 Immunocytochemistry for oxidative burst molecules in MS lesions: ................44

4 Discussion .....................................................................................................................53

References…………………………………………………………………………………………………………………………. 54

Acknowledgements…………………………………………………………………………………………………………….65

List of publications…………………………………………………………………………………………………………….. 67

Curriculum vitae………………………………………………………………………………………………………………… 68

iii

Abstract

Multiple Sclerosis is a chronic inflammatory disease of the central nervous system,

associated with demyelination and neurodegeneration. The mechanisms of tissue injury are

poorly understood, but recent data suggest that mitochondrial injury may play an important

role in this process. Mitochondrial injury can be triggered by reactive oxygen and nitric oxide

species, and we recently provided evidence for oxidative damage of oligodendrocytes and

dystrophic axons in early stages of active multiple sclerosis lesions. In this study we

identified potential sources of reactive oxygen and nitrogen species through gene expression

in carefully staged and dissected lesion areas and by immunohistochemical analysis of

protein expression. Genome wide microarrays confirmed the profound mitochondrial injury

in active multiple sclerosis lesions, which may serve as an important source of reactive

oxygen species. In addition, we found profound differences in the gene expression levels of

various nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase -subunits between

initial multiple sclerosis lesions and control white matter. These results were confirmed at

the protein level by means of immunohistochemistry, showing up-regulation of the subunits

gp91phox, p22phox, p47phox, NOX1 and NOXO1 in activated microglia in classical active as

well as slowly expanding lesions. The subunits gp91phox and p22phox were constitutively

expressed in microglia and were up-regulated in the initial lesion. In contrast, p47phox,

NOX1 and NOXO1 expression was more restricted to the zone of initial damage or to lesions

from patients with acute or early relapsing/remitting multiple sclerosis. Double labeling

showed co-expression of the NADPH oxidase subunits in activated microglia and infiltrated

macrophages, suggesting the assembly of functional complexes. Our data suggest that the

inflammation associated oxidative burst in activated microglia and macrophages plays an

important role in demyelination and free radical-mediated tissue injury in the pathogenesis

of multiple sclerosis.

iv

Zusammenfassung

Multiple Sclerose (MS) ist eine enzündliche Krankheit des Zentralnervensystem (ZNS) und

geht mit Neurodegeneration und dem Verlust von Myelinscheiden einher. Die

pathologischen Veränderungen im ZNS sind komplex und weisen je nach Individuum,

Stadium der fortschreitenden Krankheit und Art/Beschaffenheit des Entzündungsprozesses

ein breites Spektrum auf. Die Mechanismen, die zur Gewebeschädigung führen, sind

weitgehend unbekannt; neueste Forschungsergebnisse lassen aber vermuten, dass eine

Schädigung der Mitochondrien im Krankheitsprozess eine wesentliche Rolle spielt. Es ist

daher von großer Bedeutung für das Verständnis um die Ausbildung und das Fortschreiten

der Krankheit, die Ursache solcher mitochondrialen Schäden aufzuklären.

Ein Grund für ausufernden mitochondrialen Schaden sind anhaltend erhöhte Mengen an

Sauerstoff- und Sticksoff- Radikalen, und erst kürzlich konnten wir oxidative Veränderungen

an Oligodendrozyten und dystrophischen Axonen in aktiven Zonen von MS Lesionen

nachweisen. In der hier vorliegenden Studie tragen wir zur Aufklärung möglicher

Mechanismen der mitochondrialen Schädigung bei, indem wir eine weitere Quelle für

Sauerstoff- und Sticksoff- Radikale identifizieren. Gen-Expressions-Analysen

unterschiedlicher Aktivitätszonen von MS Lesionen haben profunde Unterschiede im

Expressionslevel von Molekülen, die an oxidativem Schaden beteiligt sein können, zu Tage

gebracht. Besonders große Unterschiede zwischen ungeschädigter weißer Substanz und

Zonen aktiver Gewebeschädigung wurden bei mehreren Untereinheiten der NADPH-Oxidase

festgestellt. Mit immunohistochemischen Methoden konnten wir in aktiven Zonen von MS

Lesionen erhöhte Protein-Level der Untereinheiten gp91 phox, p22phox, p47phox, NOX1

und NOXO1 nachweisen. Doppelfärbungen konnten die Ko-Expression der einzelnen

Untereinheiten auf Microglia-Zellen und perivaskulären Makrophagen zeigen, was die

Zusammensetzung eines funktionellen NADPH-Oxidase Komplexes nahelegt. Unsere Daten

zeigen, dass Mikroglia und Makrophagen durch die NADPH-Oxidase mediierte Produktion

oxidativer Radikale maßgeblich an dem entzündunglichen Prozess der Gewebschädigung im

ZNS von MS Patienten beteiligt sind.

Introduction

1

1 Introduction

1.1 Multiple Sclerosis and its pathology

Multiple Sclerosis (MS) is the most common neurological disease in young adults in the

Western world (Noseworthy et al., 2000), showing a prevalence of about 1:1000 in the

western world (Compston, 2006). The clinical course of MS is unpredictable and highly

variable, it was classified into 4 different clinical courses: relapsing-remitting MS (RRMS),

progressive-relapsing MS (PRMS), secondary progressive MS (SPMS) and primary progressive

MS (PPMS) (Lublin, 1996) (Fig. 1).

Figure 1: Clinical courses of MS: (A) Relapsing-remitting MS is characterized by clearly defined acute attacks with full recovery or with sequelae and residual deficit upon recovery. Periods between disease relapses are characterized by lack of disease progression. (B) Progressive-relapsing MS shows progression from onset but with clear acute relapses with or without full recovery. (C) Secondary progressive MS begins with an initial RR course, followed by progression of variable rate that may also include occasional relapses and minor remissions. (D) Primary progressive MS is characterized by disease showing progression of disability from onset, without plateaus or remissions or with occasional plateaus and temporary minor improvements. Figure and legend modified from Lublin et al, 1996.

Introduction

2

Initially, MS was considered a disease of the white matter and defined as an inflammatory

process, associated with focal plaques of primary demyelination in the white matter of the

brain and spinal cord (Charcot 1880). Meanwhile, four different patterns of white matter

demyelination were identified in RRMS (Lucchinetti et al., 2000) (Fig.2). Basically all patterns

involve T-cell infiltration, microglia activation and demyelination. Pattern I lesions are

thought to be mediated by macrophage toxins, pattern II lesions are characterized by

antibody/complement deposition, pattern III lesions display hypoxic changes similar to those

seen in stroke lesions, and pattern IV lesions seems to evolve on a background of a

genetically determined, increased susceptibility to immune-mediated injury (Lucchinetti et

al., 2000).

Figure 2: Illustration of the four different pathological lesion patterns in MS. On the general background of T-cell mediated inflammation, demyelination may be induced by macrophages (M) and/or their toxic products (resulting in pattern I), by specific demyelinating antibodies and complement (C, resulting in pattern II), by degenerative changes in distal processes, followed by apoptosis (pattern III). Pattern III are similar to hypoxic/ischemic white matter lesions. Finally, there can be a primary degeneration of oligodendrocytes followed by myelin destruction (resulting in pattern IV). This lesion type is characterized by demyelination and tissue damage due to a genetically determined increased susceptibility of the brain tissue for immune-mediated injury. Figure and legend taken from (Lassmann, 2001; Lucchinetti et al., 2000).

Introduction

3

However, previous studies have shown that demyelinating plaques may also be present in

the cerebral cortex (Brownell and Hughes, 1962; Peterson et al., 2001), and recent studies

revealed that the cerebral cortex can be extensively involved in MS patients, particularly in

the progressive phase of the disease (Kutzelnigg, 2005; Kutzelnigg and Lassmann, 2006) (Fig.

3).

Figure 3: Demyelination in relapsing-remitting and progressive MS: Focal inflammatory demyelinated plaques in the white matter dominate the pathology in acute and relapsing multiple sclerosis, while cortical demyelination and diffuse white matter inflammation are characteristic hallmarks of PPMS and SPMS. A, B: schematic lesion maps of multiple sclerosis brains; green: focal demyelinated plaques in the white matter; orange: cortical demyelination; Figure and legend modified from (Kutzelnigg, 2005).

1.2 The Architecture of Inflammatory Demyelinating Lesions

(Lassmann, 2011)

The classical active MS lesion:

Classical active MS lesions are most frequently found in patients with acute and relapsing

disease. During the progressive stage of the disease they become infrequent, and this is

particularly the case in patients with primary progressive MS (Frischer et al., 2009; Ingle,

2005; Kappos et al., 1999; Kutzelnigg, 2005).

The definition of classical active MS lesions is based on the presence of activated

macrophages, which contain left-overs of myelin sheaths, taken up during the demyelinating

process (Lassmann et al., 1998). Several different classifications have been proposed for the

identification of active lesions. In vitro experiments revealed a temporal sequence of myelin

degradation within macrophages, which can be used as a timeframe of lesion development

(Bruck et al., 1995; Lassmann et al., 1998). When myelin falls apart and is taken up by

macrophages, the macrophage lysosomes initially contain degradation products with all

Introduction

4

components of the intact myelin sheaths, including minor myelin proteins (myelin

oligodendrocyte glycoprotein MAG or myelin associated glycoprotein MOG). Reactivity for

these minor myelin proteins is lost within a few days after myelin ingestion, and then the

degradation products are reactive only for antibodies against major myelin proteins, such as

proteolipid protein or myelin basic protein. When these proteins are degraded about a week

after myelin ingestion, the macrophage lysosomes contain neutral lipids (stained by Oil red

O) and periodic acid Schiff reaction (PAS) positive debris. An active MS lesion, defined by the

presence of Oil red O positive or activated macrophages, which are the criteria for active

lesions in many studies on MS pathogenesis, represents a lesion stage, which persists for

months after the initial demyelinating event.

In addition, differences between acute and chronic active MS lesions have to be taken into

account. Acute MS plaques are those where macrophages, containing similar stages of

myelin digestion, are dispersed throughout the whole lesions. In contrast, in chronic active

lesions macrophages with early stages of myelin degradation are concentrated at the lesion

edge, while in the lesion center macrophages are seen with later stages of debris or

macrophages have already been removed. Even in a classical active lesion different stages of

plaque formation are present in close vicinity, which reflect different molecular mechanisms

involved in tissue damage and repair. Thus it is important to define more precisely the

architecture of active MS lesions.

1.2.1 Structural Architecture of Classical Active Lesions:

The architecture of classical active lesions is best illustrated in expanding lesions (chronic

active), but is similar also in acute plaques. When following the pathology of such lesions

from the normal appearing white matter (NAWM) into the inactive plaque core, several

distinct zones can be seen (Fig.4):

The Periplaque White Matter Area:

The normal appearing periplaque white matter adjacent to the lesion already reveals

differences to the normal white matter of controls. It contains scattered perivascular

inflammatory infiltrates, but only few lymphocytes, dispersed within the brain parenchyma

(Barnett and Prineas, 2004; Marik et al., 2007). These lymphocytes mainly consist of

CD3+/CD8+ T-cells. This is accompanied by microglia activation, reflected by the expression

Introduction

5

of CD68 in a subset of these cells. Using a general microglia marker, such as for instance Iba-

1, scattered microglia nodules are seen (Barnett and Prineas, 2004; Prineas et al., 2001),

even at some distance from the plaque. Myelin and oligodendrocytes are intact and only

exceptional axonal swellings or end bulbs are seen in sections stained for markers for fast

axonal transport. Such dystrophic axons may either reflect initial axonal injury, which

precedes demyelination, or may reflect secondary (Wallerian) degeneration of axons,

transected within the plaques.

The Initial “Prephagocytic” Lesion Area:

Approaching the edge of the lesion, microglia density increases. Myelin is still largely intact,

but frequently shows decreased staining in classical myelin stains (Luxol fast blue). In severe

and aggressive lesions a profound loss of proteins located in the most distal cell processes of

oligodendrocytes, such as myelin associated glycoprotein (MAG) and cyclic nucleotide

phoshodiesterase (CNPase) is seen (Pattern III (Lucchinetti et al., 2000)), and this is

associated with nuclear condensation and fragmentation in oligodendrocytes, suggesting

apoptotic cell death (Barnett and Prineas, 2004; Lucchinetti et al., 2000). This zone also

contains a considerable number of axonal spheroids and end bulbs, visualized by

immunocytochemistry for amyloid precursor protein (Marik et al., 2007), and these axons

are frequently surrounded by apparently normal myelin sheaths. Astrocytes reveal increased

expression of glial fibrillary acidic protein (GFAP) and in part have lost their polarity,

reflected by decreased or absent immunoreactivity of antigens, concentrated on processes

of the glia limitans, such as aquaporin 4 and α-dystroglycan (Sharma et al., 2010). This zone

of active MS lesions has been denominated as “Initial Lesions” (Marik et al., 2007) or

“Prephagocytic Lesion” (Barnett and Prineas, 2004; Henderson et al., 2009b).

The Early Active Lesion Area:

Moving further towards the centre of the lesions another zone appears. There myelin is

already lost and myelin debris is taken up by densely packed macrophages, which contain

early stages of myelin degradation in their lysosomes (Bruck et al., 1995). In this area the

highest density of axonal spheroids or end bulbs is present (Ferguson et al., 1997; Kornek et

al., 2000; Trapp et al., 1998) and oligodendrocytes are sparse. Profoundly enlarged

“protoplasmic astrocytes” are generally present in these lesion areas, many of them having

Introduction

6

lost their polarity and aquaporin 4 expression (Sharma et al., 2010). This is the lesion area,

defined as “early active lesion” by Brück et al (Bruck et al., 1995).

The Late Active Lesion Area:

Further towards the plaque centre macrophage density decreases compared to that seen in

early active lesions. Myelin is completely lost and macrophages contain later stages of

myelin degradation (MBP or PLP only) or just contain Oil red O reactive neutral lipids (Bruck

et al., 1995). Axons are reduced in density, and some axonal spheroids or end bulbs are still

present. Interestingly, however, a subset of lesions contains a substantial number of

oligodendrocytes (Prineas et al., 1989; Raine et al., 1981), which appear to be recruited from

the pool of progenitor cells. This is associated with early stages of remyelination (Bruck et

al., 1994; Goldschmidt et al., 2009; Prineas et al., 1993). Nevertheless, fully remyelinated

shadow plaques are rare in early MS (Bramow et al., 2010; Patrikios et al., 2006). One

possible explanation for this discrepancy is that remyelination is unstable, and that

remyelinated areas preferentially become sites for recurrent demyelination (Bramow et al.,

2010).

A similar structural composition is also seen in acute plaques, although not all of the above

described zones may be present in single lesions. Instead, the entire lesion may reflect only a

single or two of the above described stages. In addition, these different zones of plaque

formation may vary in size, which presumably reflects the speed of lesion development or

their stage of evolution.

Introduction

7

Figure 4: Lesional stages on the example of slowly expanding MS lesions: a-e: Slowly expanding lesion in a patient with progressive MS stained for macrophages and microglia (CD68). In the periplaque white matter (a: PPWM and b) profound microglia activation with some microglia nodules are seen. At the active edge (a: Initial/EA)) there is a small zone of further increase of microglia density and microglia activation (c) followed by a zone with scattered macrophage like cells (d). The lesion centre (Demyelinated Plaque) is hypo cellular; in particular nearly all macrophages or microglia cells have vanished from the lesion (e). The position of Figures b-e are indicated in brackets in a. f-j: The same lesion stained for myelin (Luxol fast blue; LFB and PAS); at the lesion edge few macrophages are seen, which contain early and late myelin degradation products (g, h), which are also reactive for proteolipid protein (PLP; i); in addition some dystrophic axons are seen at the lesion edge in sections stained for neurofilament (NF; j); Figure and legend modified from (Lassmann, 2011).

1.2.2 Structural architecture of slowly expanding lesions (SEL):

Slowly expanding lesions are predominantly and frequently seen in patients with progressive

MS and are also frequent in primary progressive MS (Frischer et al., 2009; Kutzelnigg, 2005).

The Periplaque White Matter Area:

Similarly as in classical active lesions SELs are surrounded by periplaque white matter with

microglia activation and some microglial nodules (Prineas et al., 2001). In contrast to earlier

stages of MS, however, diffuse microglia activation, associated with some myelin pallor and

diffuse axonal injury, and reactive astrocytic fibrillary gliosis is more pronounced in the

Introduction

8

normal appearing and periplaque white matter in progressive MS compared to early MS

(Allen et al., 2001; Kutzelnigg, 2005).

Lesion rim Area:

At the border of these lesions a narrow zone of profound microglia activation is seen. Some

macrophages are present as well, containing earliest stages of myelin degradation in their

lysosomes. Acute axonal injury is quite prominent in these areas, reflected by axonal

spheroids or end bulbs with accumulation of proteins transported by fast axonal transport.

Lesion Center Area:

Towards their centre demyelination is associated with complete loss of oligodendrocytes

and profound reduction of axonal density (Mews et al., 1998). Similar to classical active

lesions thin axons are preferentially destroyed, while thick axons largely remain preserved

(Mahad et al., 2008a). In contrast to classical active lesions there is the profound loss of

microglia and macrophages, even in areas closely adjacent to the zone of active tissue injury.

Considering the long lasting persistence of macrophages in classical active lesions this

observation suggests that macrophages and microglia are also destroyed in the active zone

of tissue damage. Reappearance of new oligodendrocytes and remyelination is sparse

(Prineas et al., 2001), although oligodendrocyte progenitor cells may still be preserved in

variable numbers (Chang et al., 2002). Overall, the centre of SELs is characterized by a dense

astrocytic scar, demyelination, profound axonal loss and decreased global cell density due to

nearly complete loss of oligodendrocytes, macrophages and microglia. In addition, ongoing

axonal degeneration, indicated by the presence of amyloid precursor protein reactive axonal

spheroids or end bulbs, is seen even within the otherwise inactive lesion centre (Kornek et

al., 2000). These structural features of SELs suggest that the mechanisms of tissue injury are

at least in part different from those seen in classical active plaques.

Introduction

9

1.3 Inflammation as a source for reactive oxygen intermediates

(Modified from (van Horssen et al., 2011))

1.3.1 Reactive oxygen species (ROS) formation and detoxification

Reactive oxygen species (ROS) are molecules which are highly reactive due to the presence

of unpaired electrons or their ability to give rise to new free radicals. ROS exist in many

different forms, with superoxide (O2−) and its derivatives hydroxyl radical (OH∙) and

hydrogen peroxide (H2O2) being most abundant in eukaryotic cells. Physiological

concentrations of ROS regulate various biological mechanisms, e.g. ventilation control and

erythropoietin production (Droge, 2002), but-in much higher local concentrations-are

produced by immune cells in order to kill pathogens (Forman and Torres, 2001; Hampton et

al., 1998). Superoxide can be formed either enzymatically by e.g. NADPH oxidase in the

plasma membrane of macrophages and microglia cells, and non-enzymatically as a by-

product of oxidative phosphorylation in the mitochondrial membrane (Cadenas et al., 1977).

Superoxide has a short life span and cannot directly attack DNA, proteins or lipids (Halliwell

and Gutteridge, 1999). It is either rapidly converted by superoxide dismutases (SODs) into

hydrogen peroxide (Deby and Goutier, 1990), or non-enzymatically converted into other ROS

(Fridovich, 1978). Hydrogen peroxide is relatively stable and can travel further distances than

superoxide. It has the potential to generate reactive hydroxyl radicals through interactions

with iron or copper in cells by means of the Fenton reaction. Superoxide can also react with

nitric oxide (NO), which is enzymatically produced by nitric oxide synthase (NOS), to form

peroxynitrite (ONOO−) (Fig.5).

Figure 5: Schematic overview of ROS formation and detoxification. Superoxide (O2.−

) is dismutated into hydrogen peroxide (H2O2) by superoxide dismutases, however in combination with nitric oxide (NO) forms peroxynitrite (ONOO.). Subsequently, hydrogen peroxide is either detoxified by endogenous antioxidant enzymes or in the presence of transition metals converted into highly toxic hydroxyl radicals (OH·). Figure and legend taken from (van Horssen et al., 2011)

Introduction

10

In the pathogenesis of MS, high amounts of ROS are produced which appear to overwhelm

the antioxidant capacity, resulting in oxidative stress. Oxidative stress manifests in ROS-

induced damage to biological macromolecules such as polyunsaturated fatty acids (PUFA) in

membrane lipids, essential proteins, and DNA/RNA. The CNS exhibits high oxygen

consumption and is enriched in PUFA, making it particularly vulnerable to lipid peroxidation.

Increased levels of indicators of oxidative stress and/or decreased levels of antioxidant

enzymes and antioxidants have been detected in blood and cerebrospinal fluid of MS

patients during the active phases of disease (Calabrese et al., 1994; Ferretti et al., 2005;

Gilgun-Sherki et al., 2004; Greco et al., 1999; Karg et al., 1999; Koch et al., 2006; van

Meeteren et al., 2005)

1.3.2 Activated microglia and macrophages as sources of ROS

Both activated microglia and infiltrated macrophages are able to generate vast amounts of

proinflammatory mediators and oxidizing radicals, such as superoxide, hydroxyl radicals,

hydrogen peroxide and nitric oxid (Colton and Gilbert, 1993). There are three major ROS-

generating enzymes found in activated microglia and macrophages: myeloperoxidase,

xanthine oxidase and NADPH oxidase. Myeloperoxidase is a lysosomal enzyme which

produces hypochlorous acid (HOCl) from hydrogen peroxide and chloride anion. Gray and

colleagues showed that demyelination was associated with significantly elevated

myeloperoxidase activity in homogenates of MS white and gray matter (Gray et al., 2008a;

Gray et al., 2008b). Immunohistochemistry revealed that myeloperoxidase was

predominantly expressed by macrophages and activated microglia within and in close

vicinity of MS plaques in white matter lesions. More recent studies showed that

myeloperoxidase was expressed by a subset of activated microglia in cortical lesions which

lack significant macrophage infiltration but contain a rim of activated microglia cells (Bo et

al., 2003; Bo et al., 2006), suggesting that microglia-derived ROS might also contribute to

gray matter demyelination (Gray et al., 2008b).

Xanthine oxidase catalyzes the oxidation of hypoxanthine to xanthine, generating large

quantities of superoxide.

NADPH-Oxidase is a multi-subunit enzyme complex that is assembled in the plasma

membrane as well is in the phagolysosomal membrane of microglia cells and macrophages

under pathological conditions and catalyzes the production of superoxide from oxygen

(Fig.6).

Introduction

11

Figure 6: NADPH-Oxidase complexes. Both NADPH-complexes convert molecular oxygen to superoxide. They differ in their catalytic, membrane bound subunit (NOX1or NOX2) and their intracellularly assembled subunits, but share the membrane bound p22phox molecule. Figure taken from http://www.med.monash.edu.au/pharmacology/research/nox-link.html

In Alzheimer’s disease, NADPH oxidase appears to mediate neuronal damage (Block, 2008),

but nothing is known about its expression pattern in MS tissue (van Horssen et al., 2010).

Under pathological conditions, microglia and macrophages are not only able to produce

large amounts of superoxide via the ROS generating enzymes explained above, but they

additionally generate high levels of nitric oxide by activation of inducible nitric oxide

synthase (iNOS). Nitric oxide reacts with superoxide to form highly toxic peroxynitrite, which

is in turn able to cause oxidative damage.

1.3.3 Oxidative phosphorylation as a source of ROS

Besides activated macrophages and microglia cells, there is second major source of reactive

oxygen species: the mitochondrial respiratory chain. Located in the inner mitochondrial

membrane, it is a constant source of ROS, due to electron leakage from the electron transfer

chain (Boveris and Chance, 1973; Cadenas et al., 1977). Normally, the amount of ROS

produced by mitochondria is counteracted by local antioxidant enzymes, however, the ROS

leakage was reported to increase under conditions of inflammation (Nathan and Ding, 2010).

1.3.4 Mitochondrial injury and neurodegeneration

The involvement of the mitochondrion in ATP metabolism as well as in oxygen consumption

have made them particularly interesting in neurodegenerating diseases and mitochondrial-

derived ROS are believed to play a major role in neurodegeneration, as previously observed

in Alzheimer's and Parkinson's disease (Beal, 2003; Lin and Beal, 2006; Moreira et al., 2008).

Nevertheless, one should keep in mind that the mitochondrion itself is not only a potent

Introduction

12

keyplayer in the production of ROS, but can subsequently also be affected by ROS induced

damage:

In vitro data provide evidence that mitochondrial injury can be induced by reactive oxygen

and nitrogen (RNI) species (Bolanos et al., 1997a; Bolanos et al., 1997b; Higgins et al., 2010;

Witte et al., 2010). Mitochondrial injury in active MS lesions mainly affects complex IV of the

respiratory chain and is seen in axons, oligodendrocyte as well as in astrocytes (Mahad et al.,

2008b). The acute mitochondrial injury, present in active lesions, is followed by a

compensatory increase in mitochondrial density and enzymatic activity in chronic

established lesions (Mahad et al., 2009). Mitochondrial injury in MS lesions has different

consequences, depending upon the affected cell type:

In axons its main effect is energy deficiency, leading to disturbance of axonal transport and

demise (Dutta et al., 2006; Mahad et al., 2009): upon demyelination, axonal conduction is

blocked due to the absence of sodium channels in acutely demyelinated segments. However,

axonal conduction can be restored by redistribution of sodium channels along these

demyelinated segments (Craner et al., 2004). This leads to an enormous influx of sodium,

which can be counteracted by increasing Na+/K+ ATPase activity. Na+/K+ ATPase function

depends on ATP, thus mitochondria in demyelinated axons have an increased need in ATP

production to maintain conduction. In a situation of energy deficiency due to impairment of

ATP synthesis, the increased energy need of the Na+/K+ ATPase turns out to be deleterious.

The consequent axonal loss affects thin axons more severely than thick ones, most likely

because thin axons have relatively less volume to surface area or ‘energy to ions’ ratio

compared with large diameter axons ((DeLuca et al., 2004; Waxman, 2006).

In oligodendrocytes, mitochondrial injury is associated with cell apoptosis, apparently

mediated through liberation of apoptosis inducing factor (AIF) from mitochondrial stores

(Veto et al., 2010).

Astrocytes and oligodendrocyte progenitors are more resistant to mitochondrial injury.

These cells survive but have functional deficiencies, such as for instance the inability to form

and sustain cell processes (Sharma et al., 2010). This may explain in part the lack of

remyelination in MS lesions, despite the presence of oligodendrocyte progenitor cells

(Ziabreva et al., 2010).

Introduction

13

1.3.5 Oxidative damage in Multiple Sclerosis lesions

The occurrence of widespread oxidative damage in demyelinating MS plaques is supported

by the expression of markers of oxidative damage in MS brain tissue. Recent data support

variant forms of oxidative damage, affecting different cell types:

Oxidative damage to proteins is reflected by nitrotyrosine residues, generated by the

diffusible free radical peroxinitrite that can react with tyrosine residues in proteins.

Nitrotyrosine residues are present in foamy macrophages and large hypertrophic astrocytes

throughout active demyelinating MS lesions suggesting that both macrophages and

astrocytes are a primary target of oxidative damage (Cross et al., 1998; Diaz-Sanchez et al.,

2006; Liu et al., 2001; van Horssen et al., 2008).

Oxidative damage to nucleotides in the DNA is mirrored by the occurrence of 8-hydroxy-2’-

deoxyguanosine (8-OHdG) (Lu et al., 2000; van Horssen et al., 2008). It was mainly seen in

oligodendrocyte nuclei, which in part showed signs of apoptosis. In addition, a small number

of reactive astrocytes revealed nuclear expression of 8-OHdG (Haider et al., 2011) (Fig.7A).

Oxidative damage to cellular membrane lipids can be followed up with three different

markers: 4-hydroxy-2-nonenal (4-HNE), malondialdehyde (MDA-2) and oxidized phospholipid

epitopes. 4-HNEis a highly reactive aldehyde that is toxic to CNS cells. In active demyelinating

MS lesions 4-HNE accumulates in both phagocytic macrophages and large hypertrophic

astrocytes (van Horssen et al., 2008).

MDA-2 is a reactive aldehyde that is formed upon oxidative damage to poly-unsaturated

fatty acids (PUFA) (Welch et al., 1997). In MS lesions it was described in the cytoplasm of

oligodendrocytes and some astrocytes (Haider et al., 2011) (Fig.7C)

Another marker for lipid peroxidation is E06 (Qin et al., 2007). It was found in the cytoplasm

of oligodendrocytes and some astrocytes as well, but in contrast to MDA-2, E06 was

massively accumulated in a fraction of axonal spheroids with disturbed fast axonal transport

as well as in neurons within grey matter lesions. Neurons stained for oxidized phospholipids

frequently revealed signs of degeneration with fragmentation of their dendritic processes

(Haider et al., 2011) (Fig.7B). Taken together, there is ample evidence that extensive

oxidative injury occurs in MS plaques and that ROS-induced damage already occurs in the

earliest stages of neuroinflammation.

Introduction

14

Figure 7: Oxidative damage in MS lesions: (A) Oxidized DNA in multiple sclerosis lesions, visualized by immunocytochemistry for 8-hydroxy-D-guanosine (8-OHdG): 1. Double staining for oxidized DNA (8-hydroxy-D-guanosine; blue) and the oligodendrocyte marker TPPP-p25 (red) at the lesion edge of an active lesion of acute multiple sclerosis. One of the six oligodendrocytes shows blue 8-hydroxy-D-guanosine reactivity in a nucleus with nuclear condensation and fragmentation (apoptotic cell). 2. Double staining for oxidized DNA (8-hydroxy-D-guanosine, blue) and glial fibrillary acidic protein (GFAP, red) at the edge of an active lesion of acute multiple sclerosis. One astrocyte contains a blue nucleus, indicating oxidized DNA. 3. Double staining for oxidized DNA (8-hydroxy-D-guanosine, blue) and the T-cell marker CD3 (red) in an active lesion of acute multiple sclerosis. No DNA oxidation is seen in the CD3-positive T-cell population. – 4. Double staining for oxidized DNA (8-hydroxy-D-guanosine, blue) and the macrophage marker CD68 (red) in an active lesion of acute multiple sclerosis. The macrophage contains a nucleus with oxidized DNA, possibly representing phagocytosis of an apoptotic cell. (B) Oxidized phospholipids in multiple sclerosis lesions, visualized by immunocytochemistry for E06: 1. Oxidized phospholipids are selectively accumulated within dystrophic axons and axonal spheroids. 2. The nature of dystrophic axons is documented by double staining confocal microscopy for oxidized phospholipids and synaptophysin. 3. Active multiple sclerosis lesions; double staining for the oligodendrocyte marker TPPP-p25 (red) and E06 (black/brown), showing one of the two oligodendrocytes with intense cytoplasmic E06 immunoreactivity. 4. In astrocytes, oxidized phospholipids are sequestered in the cytoplasm in the form of larger granules, possibly representing autophagic vacuoles. (C) MDA2 immunoreactivity in oligodendrocytes and myelin in active multiple sclerosis lesions: 1, 2. The nature of the cells as oligodendrocytes is shown by double staining using TPPP-p25 as a marker. 3. Granular cytoplasmic MDA2 immunoreactivity (blue) in the cytoplasm of a glial fibrillary acidic protein (GFAP)-positive astrocyte (red) in an active multiple sclerosis lesion. Figure and Legend taken and modified from (Haider et al., 2011)

Material and Methods

15

2 Material and Methods

2.1 Human autopsy tissues

(Fischer et al, 2011)

Our study was performed on autopsy brains of patients and control cases from paraffin

blocks archived in the Centre of Brain Research, Medical University of Vienna, Austria and

the Dept. of Neuropathology, University Medical Center Amsterdam, The Netherlands. The

MS sample from Vienna (total n=28; female to male ratio: 19/10; age range: 20 to 84)

contained 7 cases of Marburg’s type of acute MS, two of them with Balo type concentric

lesions, 3 cases of relapsing remitting MS, 10 cases of secondary progressive MS and 8 cases

of primary progressive MS. As control we included autopsy tissues from patients without

neurological disease and without any CNS lesions (n=18; female to male ratio 11/7; age

range 30 to 97). Furthermore 7/8 cases of MS with aggressive acute or relapsing disease

course, analysed in our present sample, were also included previously in our studies on

mitochondrial injury (Mahad et al., 2008b) and on initial MS lesions (Marik et al., 2007). The

samples from Amsterdam (total n=11; female to male ratio: 8/3; age range: 45 to 80)

contained 10 cases of secondary progressive MS and 1 case of primary progressive MS.

Detailed clinical data on these patients have been recently published (van Horssen et al.,

2010).

Case no Disease Type Age Gender Disease course Lesion Type

270-99-2 AMS 1 45 Male Monophasic Active (Pattern III); Inactive

S403-97-1 AMS 2 45 Male Monophasic Active (Pattern III), Inactive

Y269-99-1A AMS 3 52 Male Monophasic Active (Pattern II), Inactive

581-96-10D AMS 4 35 Male Monophasic Active (Pattern II; Inactive

70-93-3,6 AMS 5 78 Male Monophasic Active (Pattern III); Inactive

A01-144 AMS 6 34 Female Monophasic Active (Pattern III); Inactive

46-89-3C AMS 7 51 Female Monophasic Active (Pattern II), Inactive

Spanien C2 RRMS 1 40 Female RRMS Active (Pattern III); Inactive

172-81-4 RRMS 2 57 Female RRMS Active (Pattern II); Inactive

499-97-3,5 RRMS 3 20 Female RRMS Active

Y67-05-9 SPMS 1 34 Male SPMS with attacks Inactive

110-01-3 SPMS 2 62 Female SPMS without attacks Active (Pattern I)


16

423-89-5 SPMS 3 61 Female SPMS with attacks

Slowly Expanding Active;

Inactive

131-95-3 SPMS 4 56 Male SPMS without attacks Active (Pattern I); Inactive

Y243-06-6 SPMS 5 46 Female SPMS with attacks


Inactive

323-82-3 SPMS 6 84 Female SPMS without attacks Inactive3

285-81-6 SPMS 7 78 Female SPMS Inactive

72-83-6 SPMS 8 63 Female SPMS without attacks Inactive

88-91-2 SPMS 9 76 Male SPMS without attcks Inactive

9-04-28 SPMS 10 66 Female SPMS with attacks Inactive

14-90-2 PPMS 1 55 Female PPMS


Inactive

39-03-4 PPMS 2 67 Male PPMS Active (Pattern I), Inactive

267-93-15 PPMS 3 53 Male PPMS


Inactive

144-90-3,7 PPMS 4 77 Female PPMS


Inactive



Inactive



Inactive

418-83-5 PPMS 7 75 Female PPMS Inactive

55-96-7 PPMS 8 83 Female PPMS Inactive

45-05-3 Control 1 30 Female Normal Control -


162-02-4 Control 3 37 Male Normal Control -



14-04-5,7 Control 6 45 Female Normal Control -

579-91-1,3 Control 7 46 Male Normal Control -


425-92-5 Control 9 48 Male Normal Control -

J132-92-1,4 Control 10 65 Male Normal Control -

J341-92-4 Control 11 70 Male Normal Control -


J50-93-2 Control 13 72 Male Norrmal Control -

J68-93-4 Control 14 83 Male Normal Control -

J119-92-3 Control 15 84 Female Normal Control -

91-07-D Control 16 - -Female Normal Control -



Table 1


17

For microarray studies 3 of the acute MS cases (270-99-2, A01-144, S403) were selected on

the basis of lesion size and activity as well as mRNA preservation, assessed by in situ

hybridization for proteolipid protein mRNA.

2.2 Immunohistochemistry

Immunohistochemistry (IHC) uses antibodies (so called primary antibodies) directed against

specific targets for their visualization.

A common technique described below is the ABC-System (avidin biotinylated enzyme

complex).

Oxidized phospholipids (antibody E06) were stained by a special protocol (described in

2.2.2).

As substrates for visualization of secondary antibodies conjugated to horse radish

peroxidase (POX), either diaminobenzidine (DAB) (K3467 Dako®) or 3amino-9ethylcarbazole

(AEC) (K3464 Dako®) were used.

As substrates for visualization of secondary antibodies conjugated to Alkaline Phosphatase

(AP), the diazonium salt Fast blue (FB) was used.

2.2.1 IHC with ABC-System

Expression of the NADPH-subunits p22phox, gp91phox, p47phox, NOX1 and NOXO1 in

different stages of MS lesions was studied with immunohistochemistry on paraffin sections

according to established techniques (Bauer et al., 2007; King et al., 1997).

The investigations were performed on 3-5 μm thick paraffin sections of human material.

Sections were deparaffinized twice with Xylol for 2 times 15 min, rinsed twice in 96% EtOH,

treated with hydrogen peroxide in methanol (150ml methanol substituted with 1ml H2O2)

for 30 min to block endogenous peroxidises, rehydrated in a descending series of EtOH

(96%>70%>50%>30%). For some of the antibodies, antigen retrieval was performed by

heating the sections for 60-90 min in EDTA (1mM EDTA, 10mM Tris, pH 8.5 or 9) or 0.1mM

citrate buffer (pH 6) in a household food steamer device. Sections were further incubated for

1 hour in DAKO buffer (DAKO wash buffer, code S 3006) containing 10% fetal calf serum

(Cambrex)(DAKO/FCS) in a humid chamber at room temperature to block nonspecific

antibody reactions. The respective primary antibodies (listed in table 2) were applied


18

overnight in DAKO/FCS at 4°C. Afterwards, the slides were washed 3–4 times in tris buffered

saline (TBS). Then the slides were incubated with biotinylated secondary antibodies for 1

hour at room temperature. After washing 3–4 times in TBS, the sections were treated with

avidin peroxidase (diluted 1:100 in DAKO/FCS), and incubated for 1 hour at room

temperature. For visualization of the bound antibodies, diaminobenzidine (DAB) was used as

chromogen as described (Kutzelnigg, 2005). For some antibodies, Copper sulfate (2%copper

sulphate in 0,9% NaCl) treatment was additionally performed to enhance signal sensitivity.

Counterstaining of nuclei was done by rinsing in haemalum for 10 seconds, followed by a

washing step with water, a differentiation step in 0,5% hydrochloric acid alcohol, rinsing in

water and blueing in Scott’s reagent for 5 minutes. After that, the sections were washed

again in water and dehydrated in ascending series of EtOH (50%, 70%, 3 times 96%) followed

by n-butylacetate. Sections were finally mounted with Eukit.

2.2.2 IHC for Oxidized Phospholipids

The section on glass slides were deparaffinized with Xylol for 2x 20 min, rehydrated (2x 96%

Ethanol, graded series of alcohol 1x 70%, 1x 50%, A. dest.) and washed with Phosphate

Buffer(PB)/Tween20 (= Sörensen Phosphate Buffer 0,2M + 0,005% Tween20 (S1966 Dako®)).

Unspecific background reactions were blocked by 20 minutes incubation with Dako-Diluent

(S2022). After washing with PB, Avidin was blocked for 10 min by incubation with Avidin-

Block 1:100 (=0,001% in PB) followed by 10 min Biotin-Block 1:10 (=0,001% in PB) (Avidin-

Biotin-blocking system Dako® X0590). The slides were rinsed with PB and the primary

antibody was added (diluted in Dako diluent, incubated at 4°C over night). After washing off

the primary antibodies with PB/Tween20, endogenous peroxidase was blocked by

incubation with 3%H2O2 in PB for 10 min. After rinsing with PB/Tween20, biotinylated

secondary antibodies were applied (30 min at room temperature). Sections were washed

with PB/Tween20 and incubated with avidin conjugated horse radish peroxidases (HRP)

complex from Sigma® (1:100) for 30 min. DAB (K3467 Dako®) was used as chromogen. The

reaction was stopped with deionized H2O2 and counterstaining was performed with Mayer’s

alum hematoxylin, followed by washing with deionized H2O2 and dehydration through a

graded series of alcohol (50%, 70%, 96%, 96%, 96%, Xylene). Then sections were mounted

with cover slips using mounting medium Eukit.


19

2.2.3 Klüver-Barrera- PAS

Klüver-Barrera-PAS stains myelin-sheaths in blue and grey matter in red. This staining was

perfomed for every case as orientation-help.

Sections were deparaffinized twice with Xylol for 2 times 15 min and rinsed twice in 96%

EtOH. Afterwards, the slides were incubated in 0,1%Luxol Fast Blue at 50-60°C over night.

After cooling and washing in 96% alcohol and distilled H2O, sections were incubated in 0,1%

lithiumcarbonate for 5 min. Differentiation was done in 70% EtOH until only myelin sheath

remain stained, followed by washing with dH2O and incubation in 0,8% periodsäure for 10

minutes. Afterwards, sections were rinsed with dH2O, put to Schiff’s reagent for 20 min,

incubated 3 times for 2 min in sulphite washing solution, dehydrated (ascending series of

EtOH followed by n-butylacetate) and mounted with Eukit.

2.2.4 Markers used

Primary antibodies are listed below (Table 2)

Primary Antibody Antibody Type Target Dilution Pretreatment Source

E06 Mouse (mAB) Oxidized

phospholipids

1:234 - Courtesy of Dr.

Binder

PLP Mouse (mAB) Proteolipid Protein, 1:1000 EDTA pH9 Serotec, MCA 839G

CD3 Mouse (mAB) T-cell antigen 1:50 EDTA pH9 Biocare

CD8 Mouse (mAB) T-cell antigen 1:100 EDTA pH9 Dako

CD20 Mouse (mAB) B-cell antigen 1:100 EDTA pH9 NeoMarkers

CD68 Mouse (mAB) Macrophages,

microglia

1:100 EDTA pH 9 Dako, M0814

HLA-DR Mouse (mAB) MHC II molecules 1:100 Citrate pH6 Dako

iNOS Rabbit (pAB) Inducible nitric

oxide synthase

1: 30.000 EDTA pH9 Chemicon

Iba-1 Rabbit (pAB) Macrophages,

microglia

1:3000 EDTA pH9 Courtesy of Dr. Sonja

Fors-Petters

NOX1 Rabbit (pAB) NADPH-Oxidase

subunit

1:200 EDTA pH 9 Sigma SAB200097

gp91phox Mouse (mAB) NADPH-Oxidase

subunit

1:100 Citrate pH6 Verhoeven et al 1989

p22phox Rabbit (pAB) NADPH-Oxidase

subunit

1:100 Citrate pH6 Santa Cruz sc29781

NOXO1 Rabbit (pAB) NADPH-Oxidase

subunit

1:200 Citrate pH6 Sigma SAB1900367


20

p47 Goat (pAB) NADPH-Oxidase

subunit

1:100 Citrate pH6

Fast blue

development!

Abcam, Cambridge,

UK; ab 74095 and

Lifespan Biosci. /

Eubio, Vienna,

Austria; LS-B2365

GFAP Mouse (mAB) NADPH-Oxidase

subunit

1:200 Citrate pH6 NeoMarkers

LN3 Mouse (mAB) MHCII 1:50 Citrate pH DAKO, Glostrup,DK

CD8 Mouse (pAB) CD8 + T-cell

subpopulation

1: EDTA pH 9 DAKO

Table 2

Secondary antibodies are listed below (Table 3)

Secondary Antibodies Target Dilution Source

Donkey-bi-α-Rabbit rabbit Ig 1:2000 Jackson

Donkey-bi- α-Sheep/Goat Sheep/goat Ig 1:200 Amersham

Sheep-bi- α-Mouse Mouse Ig 1:500 Jackson

Donkey α-Mouse-POX Mouse Ig 1:100 Jackson

Donkey- α-Rabbit-POX Rabbit Ig 1:100 Jackson

Table 3

2.2.5 Double staining for light microscopy

The cellular localization of the NADPH-markers as well as the co-localization of the complex

forming subunits within the same cell type was examined with double staining for light

microscopy (p22phox, p47phox and NOXO1) and/or fluorescent microscopy (p22phox,

p47phox, gp91phox and NOX1).

All double labeling was performed using primary antibodies from different species.

Double stainings need to be performed in a way that allows the visualization of two different

primary antibodies with two chromogens of different colour on the same slice. It is required

that the two primary antibodies originate from different species.

After deparaffination, pretreatment and blocking of endogenous peroxidase (as described

above), the two different primary antibodies were applied together in Dako/10%FCS (4°C,

over night). The secondary system was chosen in a way that couples one of the antibodies

(the less delicate one) to a secondary antibody directly conjugated to a peroxidase (POX). In

the same incubation step, the signal of the more sensitive other primary antibody was

amplified by additional binding of a biotinylated second antibody. In the following step, the


21

biotinylated antibody was detected by an avidin-coupled alkaline phosphatase (AP). AP was

then first visualized by FB, resulting in a blue staining. Following the FB development, POX

was visualized with AEC, forming a red complex. Double stained structures appear violet in

the double staining. Performed double staining, the primary and secondary antibodies, their

respective dilutions and pretreatments are summarized in table 4.

Primary antibody Dilution Pretreat Secondary antibody Dilution Development

p47

+

GFAP

1:100

1:200

Citrate pH6 Donkey-bi- α-

Sheep/Goat+

Streptavidin- AP

Donkey α-Mouse-POX

1:200

1:100

1:100

Fast Blue

+

AEC

p47

+

CD68

1:100

1:100


Sheep/Goat+

Streptavidin- AP

Donkey α-Mouse-POX

1:200

1:100

1:100

Fast Blue

+

AEC

p47

+

p22phox

1:200

1:100


Sheep/Goat+

Streptavidin- AP

Donkey- α-Rabbit-POX

1:200

1:100

1:100

Fast Blue

+

AEC

NOXO1+

GFAP

1:100

1:200

Citrate pH6 Donkey-bi-α-Rabbit+

Streptavidin- AP

Donkey α-Mouse-POX

1:2000

1:100

1:100

Fast Blue

+

AEC

NOXO1

+

CD68

1:100

1:100

Citrate pH6 Donkey-bi-α-Rabbit+

Streptavidin- AP

Donkey α-Mouse-POX

1:2000

1:100

1:100

Fast Blue

+

AEC

Table 4

2.2.6 Double staining for fluorescence microscopy

3-5 μm thick paraffin sections of human material were used. The sections were

deparaffinized as described above, treated with hydrogen peroxide in methanol to block

endogenous peroxidases, and incubated for 20 min in Dako diluent to block nonspecific

antibody reactions. For some of the antibodies, antigen retrieval was performed (details

given in table 5). Then the primary antibodies were added. The primary antibodies

originating from different species were applied simultaneously overnight in Dako diluent at

4°C.

The secondary antibodies were directly coupled to a fluorophor, being Cy2, DyLight488, Cy3

or Alexa 546 and 448.


22

The signal of some delicate antibodies was enhanced by incubation with biotinylated

secondary antibody followed by incubation with a Streptavidin coupled fluorophor.

After incubation with the final secondary antibody coupled to a fluorophor, the sections

were rinsed in dH2O and mounted with Gallate-Geltol.

All antibodies used, their respective dilutions and pretreatments are listed in table 5.

Double staining of gp91phox with markers for astrocytes (GFAP), macrophages (LN3) and

other complex components (p22phox and p47phox) were performed by Jamie Lim

(Department of Molecular Cell Biology and Immunology, VU Medical Center, Amsterdam,

The Netherlands).

Primary antibody Dilution Pretreat Secondary antibody Dilution

NOX1+

GFAP

1:100

1:100

Citrate pH6 Goat- αRabbit-Cy2

Donkey- α-Mouse-Cy3

1:100

1:200

NOX1+

CD68

1:100

1:50

EDTA pH 9 Goat- αRabbit-Cy2


1:100

1:200

p22phox+

GFAP

1:50

1:100

Citrate pH6 Donkey-bi-α-Rabbit 1h+

Streptavidin-Cy3 1h

Donkey- α.Mouse

DyLight488

1:2000

1:75

1:100

p22phox+

CD68

1:50

1:50

EDTA pH9 Donkey- αRabbit-Cy2


1:100

1:200

p22phox+

CD3

1:50 EDTA pH9 Donkey-bi-α-Rabbit 1h+

Streptavidin-Cy3 1h

Donkey- α.Mouse

DyLight488

1:2000

1:75

1:100

p22phox+

CD8


Streptavidin-Cy3 1h

Donkey- α.Mouse

DyLight488

1:2000

1:75

1:100

p22phox+

CD20


Streptavidin-Cy3 1h

Donkey- α.Mouse

DyLight488

1:2000

1:75

1:100

Table 5


23

2.2.7 Quantitative analysis

Expression levels of p22phox and gp91phox in different lesion areas and the normal

appearing white matter (NAWM) were determined by densitometry, as described in detail

earlier (Haider et al., 2011). In short, different lesion areas and NAWM were defined on

sections stained with Luxol fast blue and for microglia activation (Iba-1 immunoreactivity).

From each different MS or control case 8-34 images (0.61mm/0.46mm in size) were scanned

and stored as JPEG files. The images were processed with Adobe Photoshop CS2 by setting a

threshold level (output level = 128) and pixels above this level were deleted. Percent areas,

covered by the signal, were measured with Image J. Averages for individual densitometric

values were calculated per lesion area per case and the averages compared between

different MS lesion areas and control white matter.

2.2.8 Statistical analysis

Due to the uneven distribution of the histological data, statistical analysis was performed

with non-parametric tests. Descriptive analysis included median value and range. Differences

between two groups were assessed with Wilcoxon – Mann – Whitney U – test. Differences

between more than two groups were assessed with Kruskal – Wallis test, followed by pair-

wise Wilcoxon – Mann – Whitney U – tests. In case of multiple testing (comparison of more

than two groups) significant values were corrected with Bonferroni procedure.

Interdependence of variables was evaluated by Spearman non-parametric correlation test.

The reported p-values are results of two-sided tests. A p-value smaller to 0.05 is considered

statistically significant. For all statistical analysis, mean values per patient for each lesion

type and NAWM were used. Statistical analysis was performed with IBM SPSS.


24

2.3 Whole genome arrays

2.3.1 Assessment of RNA quality: In situ hybridization

After histological characterization of the different disease cases with respect to different

inflammatory infiltrates and loss of Myelin makers, it was assessed whether the quality of

the RNA in the sample is well enough preserved to use it for array analysis.

This was done with the help of in situ hybridization.

In the in situ hybridization, a probe of antisense RNA (aRNA) is allowed to hybridize to the

complementary mRNA, thereby detecting its presence (Fig. 8).

Figure 8: Schematic illustration of in situ hybridization: A labeled probe binds to the complementary sequence on the mRNA.Figure taken and modified from www.members.cox.net

We always used the same PLP (proteolipid protein) probe for the quality assessment.

Generally, RNA that performs well in the in situ hybridization is of good enough quality for

array analysis. There seems to be a vague correlation between the time that passes until the

in situ-signal appears first and the RNA quality: the earlier a PLP-signal appears in situ, the

better the RNA quality. Nevertheless, e.g. in a very fulminant MS, a missing PLP signal in the

in situ hybridization can also be the result of the myelin loss in the lesion and does not

necessarily indicate bad RNA quality. Therefore, it is more suitable for the assessment of the

RNA quality to evaluate the in situ signal in the NAWM, were a good PLP-signal can be

expected.

Blocks which did not perform well in situ hybridization were less likely taken for RNA

isolation, blocks for which RNA hybridization did not work were excluded from array

analysis.


25

Technically, the in situ hybridization was performed as described before (Breitschopf et al.,

1992).

After deparaffination and rehydration, RNA was fixed in 4% Paraformaldehyde for 20

minutes. Sections were rinsed with Tris buffered saline (TBS), followed by protein

denaturation by incubation with 0,2M HCl. Sections were rinsed in TBS again and then tissue

was partially digested with Proteinase K (in TBS, supplemented with CaCl2) to expose the

RNA. Afterwards the slides were washed in TBS and left at 4°C for 5 minutes. To avoid non-

specific binding, the slide was incubated with 0,5% Acetanehydrid (in TBS, pH 8) for 10

minutes. Slices were dehydrated and put into a wet chamber (50°C, 30 minutes) to facilitate

the dispersial of the hybridization mix containing the proteolipid protein 1 (PLP1) probe

labeled with digoxigenin. The 1.4 kb RNA probe was constructed from a plasmid containing

the DM20 variant of the proteolipid protein 1 sequence from mouse that binds human

PLP/DM20 because of the high homology.

After application of the probe, a coverslip was put for protection and the slide was heated to

95°C for 4 minutes to linearize the RNA. Afterwards the hybridization was allowed to take

place at 65°C over night. The coverslip was removed by incubation in 2x saline sodium citrate

(SSC) followed by three highly stringent washing steps with 50% formamide in 1xSSC for 20

minutes each at 55°C. Sections were then washed three times with 1x SSC for 15 minutes,

rinsed with TBS and incubated with Boehringer Blocking Reagent containing 10% FCS for 15

minutes. Alpha-Digoxigenin coupled to Alkaline Phosphatase (solved in blocking reagent)

was applied to the slices for 1 hour. After five final washing steps with TBS, the development

was performed with NBT/BCIP at 4°C, up to 140hours. For assessment of RNA quality, the

signal appearance was controlled microscopically. Signal evaluation was performed after 26

hours of incubation (Fig.9).

We controlled for the specificity of the in situ signal with two approaches:

a) We performed in situ hybridization using sense RNA probes. Those probes were obtained

from the same plasmid but should not be able to bind to the mRNA since they are not

complementary. No signal was obtained with sense probes.

b) Microscopically we observed the appearance of the PLP signal exclusively in

oligodendrocytes, which are the only cells capable of PLP production in the the CNS.


26

Figure 9: In situ signal intensities: The strength of the in situ signal of the different blocks was compared after 26 hours of substrate reaction. Six different signal intensities were defined: A,B: no signal (-); C,D: weak signal in focal cells (+); E,F: weak signal in most oligodendrocytes (++); G,H: strong signal focally and moderate signal in most oligodendrocytes (+++); I,J: strong signal in most/all oligodendrocytes (++++); very strong signal in all oligodendrocytes (+++++); (A,C,E,G,I x32) (B,D,F,H,J x80)


27

2.3.2 Scratching of areas of interest

Sections from formaldehyde-fixed, paraffin-embedded (FFPE) tissue were used.

Consecutively cut sections of 6-10 µm were mounted on glass slides in RNAse-free

conditions. For better morphological orientation, the first section was stained with Klüver-

Barrera for visualization of Myelin sheath. Before starting the sratching, areas of interest

were figured out with the help of IHC and appropriate markers as described above. The

localization of interest was marked in the Klüver-Barrera stained slide which was used as

template for the scratching with a scalpel from the unstained slices (Fig. ). This procedure

ensured that only the area of interest was scratched for further analysis. The scratched FFPE

material was collected, each region of interest to a separate Eppendorf tube.

Figure 10: Scratching of regions of interest on the example of cortical areas: Selected regions (marked in blue) were scratched from an unstained slice. Subsequent Klüver-PAS-staining shows that the selected region was accurately separated from the rest.

2.3.3 RNA Isolation and Amplification

The RNA isolation was performed using the Roche High Pure FFPE RNA Micro Kit.

The scratched material in the Eppendorf tubes was deparaffinated with Xylol, afterwards

total RNA was isolated.


28

The mRNA in the isolate was then transcribed to the cDNA using the Paradise® Reagent

System (Arcturus, USA). This amplification system was chosen because it uses poly-T-primers

for the reverse transcription (RT) from total RNA to cDNA. This ensures a reverse

transcription starting from the poly-A tail of the total messenger RNA. In the handling of

FFPE material, this is an important point, because the RNA obtained from FFPE material is of

poor quality and prone to cross-linkage and strand breakage. As the poly-A tail protects the

RNA from 3’ degradation, the chance of transcribing well preserved mRNA is higher when

the RT starts from the poly-A tail.

The Paradise® Reagent System utilizes two round of a five step process for linear

amplification of the mRNA fraction of total cellular RNA (Fig. 11).

First-strand synthesis with poly-T-Primers linked to a T7 promotor sequence yields single

strand cDNA with an incorporated promoter sequence for T7 RNA Polymerase.

Second strand synthesis reaction yields double-stranded cDNA.

cDNA purification

in vitro transcription (IVT) with the T7 RNA Polymerase yields antisense RNA (aRNA)

aRNA isolation

Figure 11: Conversion from total mRNA to cDNA starting from the protected 3’ poly-A-tail using the Paradise® Reagent System

Two rounds of linear amplification were performed and the obtained purified aRNA was

then sent to Imagenes (imaGenes GmbH, D-13125 Berlin, www.imagenes-bio.de) where the

RNA was labeled and hybridized to whole genome Agilent platforms.

The Roche High Pure FFPE RNA Micro Kit as well as the Paradise® Reagent System was used

according to the instructions of the manufacturer.

http://www.imagenes-bio.de/


29

2.3.4 Agilent arrays

For labeling and hybridization to whole genome arrays, the amplified mRNA was sent to the

company imaGenes (imaGenes GmbH, D-13125 Berlin, www.imagenes-bio.de).

The reason for choosing the imaGenes Service was that they specialized on the hybridization

of FFPE derived RNA. The hybridization probes spotted on the arrays are designed in a way

to detect gene sequences in the 3’ end of the RNA, which is needed since the RNA was

amplified from the 3’end.

Labeling of RNA

For the Cy3 labeling, theLow RNA Input Linear Amplification Kit, PLUS, One colour was used

(Agilent p/n 5188-5339). The labelled cRNA was purified using Quiagen’s RNeasy spin

columns (RNeasy Mini Kit, Quiagen p/n 74104) and quantified using a NanoDrop ND-1000

Spectrophotometer. Labeling was performed according to the manufacturer's instructions

(One-Color Microarray-Based Gene Expression Analysis Protocol Version Feb.2007, Agilent

Technologies)

Hybridization to Agilent G4112A whole genome microarrays

The Hybridization Mix was prepared using the Agilent Gene Expression Hybridization Kit

(Agilent p/n 5188-5242) and hybridized to the Arrays using the Agilent Microarray

Hybridzation Chamber Kit (G2534A). After the hybridization, the Arrays were washed using

the Gene Expression Wash Buffer 1 (Agilent p/n 5188-5325) and Gene Expression Wash

Buffer 2 (Agilent p/n 5188-5326). The Hybridization was performed according to the

manufacturer's instructions (One-Color Microarray-Based Gene Expression Analysis Protocol

Version Feb.2007, Agilent Technologies)

Scanning of arrays

The scanning was done with the Agilent Microarray Scanner (Agilent p/n G2565BA).

Information from scan data was processed using the Agilent’s Scan Control software, version

A.7.0.1 and data were extracted with the Agilent Feature Extraction Software 9.5.1. The

scanning as well as the feature extraction was performed according to the manufacturer's

instructions (One-Color Microarray-Based Gene Expression Analysis Protocol Version

Feb.2007, Agilent Technologies)



30

Data processing

The normalization method selected for our experiments was quantile normalization (Bolstad

et al., 2003). The 75th percentile signal value was used to normalize the Agilent one-colour

microarray signals for inter-array comparisons.

The microarray data were evaluated based on log 2 fold changes in gene expression

between the sample of interest and the controls.

2.4 RNA quality control

With this FFPE tissue, several problems had to be overcome: The time-interval between

sample acquisition and fixation was unclear, it was not known whether the tissue has been

adequately cooled before fixation to prevent the action of RNA degrading enzymes, and the

tissue has been fixed with formaldehyde, which induces the formation of methylol crosslinks

(Ahlfen, 2007). This makes it essentially impossible to retrieve larger amounts of intact

mRNA but allows for the isolation of mRNA fragments of at least 480bp length.

To overcome these problems, we performed in situ hybridization with myelin-specific probes

(proteolipid protein 1 (PLP1), 1.4 kb RNA probe labeled with digoxigenin) as described above

to identify tissue blocks with good RNA preservation and we only continued with RNA

isolation procedures with tissues yielding a strong hybridization signal. Afterwards, the

different lesion areas were separated by scratching with a scalpel from unstained slides as

described earlier (Nicolussi et al 2008). The scratched material from each region of interest

was collected into separate vials. We isolated total RNA from the material, using the High

Pure FFPE RNA Micro Kit (Roche) which was specifically designed for formaldehyde-fixed

paraffin-embedded (FFPE) material. We then transcribed the mRNA fragments contained in

the total RNA pool to cDNA, using the Paradise® Reagent System (Arcturus, USA) according

to the instructions of the manufacturer. This system uses poly-T primers for the reverse

transcription from total RNA to cDNA thereby relying on the presence of the poly-A tail on

the mRNA fragments. The obtained cDNA was amplified by in vitro transcription and the

success of the RNA isolation and the suitability of the isolated material for array analysis was

tested by PCR. We designed primers which were specific for the housekeeping gene beta-

actin. The binding site of the forward primer was located 472 bases from the poly-A tail.

Only if the mRNA fragments obtained from the isolation process exceeded this length, the


31

forward primer was able to bind and a PCR product was detected and we continued with the

second round of linear amplification (Table 6)

The obtained purified aRNA was then sent to imaGenes (imaGenes GmbH, D-13125 Berlin,

www.imagenes-bio.de). The cRNA was Cy3 labeled and hybridized to Human Whole Genome

4x44 K Agilent platforms.

With the described workflow we made sure that our RNA probes had at least a length of

480bp. This was a useful size to identify many, but not all differentially expressed genes. For

example, transcripts of p22phox (CYBA) could be detected: The CYBA-oligomere spotted on

the Agilent array (A_23_P163506) binds in a distance of 407 bp from the poly-A-tail of the

CYBA gene (NM_000101.2). Accordingly, we were able to obtain corresponding signals on

the Agilent microarray. The situation was different when binding sites of oligomeres were

located outside our RNA fragment size range. For example, the PLP1-oligomere spotted on

the Agilent array (A_23_P85201) binds in a distance of 1024 bp from the poly-A-tail of the

PLP gene (NM_000533.3). Hence, such transcripts could not be detected.

PCR conditions:

Temperature Time Process Number of cycles

95°C 10 minutes initial denaturation 1

95°C

55°C

72°C

30 seconds

30 seconds

30 seconds

denaturation

annealing

elongation

40

72°C 10 minutes final elongation 1

Forward primer: 5’-TTG ACT CAG GAT TTA AAA ACT GG-3’

Distance to Poly-A tail: 472 bp

Reverse primer: 5’- AGG GAC TTC CTG TAA CAA CGC-3’

Distance to Poly-A tail: 335 bp

Length of amplified fragment: 158 bp

Table 6: Testing for RNA preservation with PCR for ß-actin (ACTB)



32

2.5 Isolation, culturing and staining of Astrocytes

The expression of the NADPH-Oxidase membrane bound subunit p22phox was also tested in

primary astrocyte cultures obtained from mixed glia cultures from the brains of newborn

Lewis rats.

2.5.1 Astrocyte-enriched cultures

Coating of cover slips

The cover slips were heated in a loosely covered glass beaker in 1M HCl at 50-60°C for at

least 4 hours. After cooling to room temperature, they were washed with ddH2O to remove

the HCl and sonicated in water bath for 30 minutes. The washing step with ddH2O and the

consecutive sonication for 30 minutes were repeated another 2 times. Afterwards, the

beaker was filled subsequently with 50%, 70%, 95% EtOH and sonicated for 30 minutes in

each EtOH dilution. The cauterized cover slips were transferred to a plastic rack and dried in

a sterile glass tray at 70°C for 2 hours. 8 cover slips were transferred to one Petri dish. A

Pasteur pipette was used to spot each coverslip with 3 parrafin dots to mark the side of the

following poly-L-lysine treatment and to serve as spacers between the cell layer that is

supposed to grow on the poly-L-lysine coated cover slip and the bottom of the plate.

The paraffin-spotted side of each cover slip was then coated with 50µl of a 1mg/ml poly-L-

lysine dilution over night at 37°C/5% CO2. After the coating, the coverslips were rinsed in

ddH2O for 5 minutes followed by 2 hours. The dishes containing the coverslips were filled

with medium and stored like that in an incubator at 37°C/5% CO2 until usage.

Flask coating

To coat the tissue flasks, 50µl Poly-L-Lysine stock (1mg/ml) were added to 10ml PBS, mixed

well and distributed evenly over the floor of the cell culture flask, which was then placed

into the incubator (37°C/5% CO2). The coated flasks could be used after 24hours of

incubation. Prior to their use, they were washed twice with 10ml PBS at room temperature.

Isolation of rat brains

The rats were decapitated and their heads were moistened with 70% ethanol for

sterilisation. The whole brain was then isolated by cutting the head above each ear and

opening the skull. The fresh brain was immediately placed in a 10ml culture dish, containing


33

10ml DMEM/Hepes for tissue conservation. After the careful and complete removal of the

meninges and blood vessels, the remainder of the brain was transferred to a fresh 10ml

culture dish containing 10ml RPMI 10% FBS medium.

Generation of mixed glia cultures

For each flask, 1 brain was processed further. The brains were triturated to generate a cell

suspension containing astrocytes, neurons, oligodendrocytes, fibroblasts, ependymal cells

and microglia cells. This cell suspension of one brain was then transferred into a Poly-L-

Lysine coated culture flask and incubated in 20ml RPMI 10%FBS for 1-2 days (37°C, 5% CO2).

After the cells of the suspension became adherent (~1-2 days), the remaining cell clumps

were removed by replacing the supernatant with new RPMI/10%FBS medium. The tissue

flasks were then again incubated at 37°C/5% CO2.

During the following 5-7 days mainly the astrocytes, fibroblasts, ependymal cells and

microglia cells survive. Astrocytes and fibroblasts form a monolayer on top of which

microglia cells and O2A progenitors start to grow. The O2A progenitor cells will give rise to

oligodendrocytes and type 2 astrocytes.

Obtaining astrocyte-enriched cultures

To remove the microglia cells and O2A progenitors which are both loosely adherent to the

monolayer, the culture flasks were tightly sealed and placed on a rocking platform (37°C,

170rpm) for at least 4 hours. Afterwards, the supernatant containing microglia and O2A

progenitor cells was discarded and the remaining monolayer was washed twice with PBS to

make sure that no medium is left over in the flask. The monolayer was trypsinised with 2ml

of Trypsin-EDTA (Sigma) for 5 minutes. The trypsinization was stopped by addition of 5ml

RPMI 10% FBS and the cell suspension was diluted 1:7 in the same medium to obtain a cell

density of about 105 cells/ml. 7 ml of this dilution were transferred into a 6mm Petri-dish

containing 8 poly-L-lysine coated cover slips. The Petri dish was put into the incubator at

37°C/5%CO2 over night to allow the adherence of the astrocytes.

Based on GFAP staining, 75% of the cells that make up the monolayer that forms on the

cover slips after that procedure are astrocytes.


34

2.5.2 Treatment of astrocyte-enriched cultures

For the treatment, each cover slip was transferred to a separate well of a 12-well-plate and

inverted. Small dots of paraffin served as spacers between the cell layer of the cover slip and

the bottom of the 12-well-plate. The astrocytes growing on the cover slip were either left

untreated as a control, or received TNFα (4ng/ml) for 12 h. Afterwards, the cover slips were

washed twice with PBS and used for immunocytochemical staining and analysis.

2.5.3 Immunocytochemical staining and analysis

The differentially treated cells were fixed with 4% PFA for 15 min (room temperature),

washed with 0.05 M Tris-buffered saline (TBS) (pH 7.4).The cells were then washed twice

with TBS, blocked with TBS containing 10%donkey serum (30 minutes, room temperature)

and incubated overnight at 4°C with commercial rabbit anti-p22phox antibody (diluted 1:100

in TBS/1% donkey serum) to detect surface p22phox expression. After washing the cells

twice with TBS they were permeabilized with TBS containing 0.5% Triton, 5% bovine serum

albumin (BSA, Sigma) and 5% donkey serum (Sigma) for 30 min at room temperature and

incubated with mouse anti-GFAP antibody (diluted 1:100 in TBS/1% donkey serum) for 1 h at

room temperature. To exclude signals resulting from unspecific binding of the secondary

fluorescence-coupled antibodies, we included negative controls which were treated in the

same way, but incubated with TBS/1% donkey serum without any primary antibody. All cells

were washed three times with TBS followed by incubation with donkey anti-rabbit Cy3

(Jackson Immuno Research Laboratories, West Grove, PA, USA)(1h, room temperature) to

visualize p22phox (red) and donkey anti-mouse Cy2 (Jackson Immuno Research

Laboratories) to identify astrocytic GFAP (green) (both diluted 1:100 in TBS/1% donkey

serum). The cells were washed with TBS and mounted with gallate/geltol. Once the cover

slips were dried (after about 24 hours, 4°C), they were analyzed by fluorescent microscopy.

For this purpose, we had a Leica TCS SP5 LASAF microscope (Leica Microsystems, CMS-

GmbH, Germany) at our disposal, equipped with an argon-ion laser [488nm excitation] and

two HeNe lasers [543nm and 633nm excitations].

Results

35

3 Results

3.1 The project

Pathological alterations in the brain of multiple sclerosis patients are complex and differ

between stages of the disease (relapsing versus progressive) or activity of the disease

process (Frischer et al., 2009; Lassmann, 2011). Active lesions consist of the classical acute or

chronic active lesions, which are characterized by profound inflammation, blood brain

barrier injury and rapidly developing demyelination and tissue injury. They are most

frequently seen in patients with acute or relapsing-remitting MS. In contrast, in patients with

progressive disease the brain contains, besides diffuse injury in the normal appearing white

and grey matter, mainly inactive lesions or slowly expanding active lesions (Kutzelnigg,

2005). Both classical active lesions and to a lesser extent slowly expanding lesions are

surrounded by a zone of microglia activation associated with initial stages of tissue injury

(Lassmann, 2011), called the initial (Marik et al., 2007) or the “pre-phagocytic” lesion stage

(Barnett and Prineas, 2004). Active lesions in the relapsing-remitting as well as in the

progressive course of MS are always associated with inflammation (Frischer et al., 2009).

Moreover, the extend of lipid and DNA oxidation correlates significantly with inflammation

(Haider et al., 2011). Recent data suggest that mitochondrial injury and subsequent energy

failure, driven by inflammation and oxidative stress, plays a major role in the induction of

demyelination and neurodegeneration (Haider et al., 2011; Mahad et al., 2008b). In vitro

data and experimental MS animal models provide evidence that mitochondrial injury can be

induced by reactive oxygen (ROS) and nitrogen (RNS) species (Bolanos et al., 1997b; Higgins

et al., 2010; Nikic et al., 2011; Witte et al., 2010). But the mitochondrion itself is not only

affected by ROS-induced damage, it is also a potent source of ROS production as disturbed

oxidative phospholylation leads to increased ROS generation (Murphy, 2009).

With the help of whole genome microarrays performed on immunohistochemically well

characterized cases and carefully selected areas, we focused on white matter injury in

relapsing-remitting MS.

Here, we concentrated on material obtained from patients who suffered from fulminant

active MS. We carefully separated 3 different lesion stages: the normal appearing white

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36

matter (NAWM), the initial damage zone and the early demyelinated lesion center.

Comparing the gene expression profile of those different lesional stages, we tried to identify

possible sources for ROS production in relation to demyelination and neurodegeneration

(“NADPH Oxidase Expression in Active Multiple Sclerosis Lesions in Relation to Oxidative

Tissue Damage and Mitochondrial Injury”).

3.2 NADPH Oxidase Expression in Active Multiple Sclerosis Lesions in Relation

to Oxidative Tissue Damage and Mitochondrial Injury

(Figures 12, 14, 15, 16, Tables 7, 8, 9, 10, 11, legends and explanations taken from Fischer et

al., 2011).

Different mechanisms might contribute to tissue injury in MS, but one of the major driving

forces was recently found to be mitochondrial damage and subsequent energy failure (Dutta

et al., 2006; Lu et al., 2000; Mahad et al., 2008b; Trapp and Stys, 2009; Witte et al., 2009;

Witte et al., 2010). Mitochondrial injury in active MS lesions mainly affects complex IV and

might explain characteristic pathological features of MS lesions, including demyelination and

oligodendrocyte apoptosis (Veto et al., 2010), destruction of small diameter axons (Mahad

et al., 2008a; Mahad et al., 2009), neurodegeneration (Campbell et al., 2011), differentiation

arrest of oligodendrocyte progenitor cells and remyelination failure (Ziabreva et al., 2010), as

well as astrocyte dysfunction (Sharma et al., 2010). In vitro data and experimental MS animal

models provide evidence that mitochondrial injury can be induced by reactive oxygen (ROS)

and nitrogen (RNS) species (Bolanos et al., 1997b; Higgins et al., 2010; Nikic et al., 2011;

Witte et al., 2010). The mitochondrion itself is not only affected by ROS-induced damage, it

is also a potent source of ROS production as disturbed oxidative phosphorylation leads to

increased ROS generation (Murphy, 2009). ROS and RNS induced damage to biological

macromolecules such as polyunsaturated fatty acids in membrane lipids, proteins, and

DNA/RNA have been described to occur in MS lesions (Cross et al., 1998; Diaz-Sanchez et al.,

2006; Liu et al., 2001; van Horssen et al., 2008). In a recent study we found profound

oxidation of DNA in oligodendrocytes, and oxidized lipids in myelin, oligodendrocytes and

axons in association with active demyelination and neurodegeneration (Haider et al., 2011).

Besides an unavoidable by-product of cellular respiration, ROS are synthesized by dedicated

enzyme systems, including myeloperoxidase, xanthine oxidase and NADPH oxidase in

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37

activated microglia and macrophages. Myeloperoxidase has been shown to be

predominantly expressed by macrophages and activated microglia within and in close

vicinity of MS plaques in white matter lesions (Gray et al., 2008a; Marik et al., 2007), as well

as in a subtype of microglia surrounding cortical lesions (Gray et al., 2008b). Expression of

nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, which convert molecular

oxygen to superoxide, has so far not been analyzed in MS lesions.

Hence the aim of our current project was to identify possible sources for ROS production in

relation to demyelination and neurodegeneration in the early stages of MS lesion

development In a first step we analyzed mitochondrial genes and those, which are involved

in oxidative stress, to obtain a global view on their expression patterns in different stages of

active lesions in three cases of fulminant acute MS in comparison to controls. In a second

step we concentrated on protein subunits of the NADPH complexes by

immunocytochemistry in a large sample of different MS lesion types and disease stages.

3.2.1 The importance of lesional staging for gene expression studies on MS material

On a structural basis MS lesions show characteristic features, which include demyelination,

loss of oligodendrocytes (Barnett and Prineas, 2004; Lucchinetti et al., 1999), preferential

destruction of thin caliber axons (Evangelou et al., 2001), impaired remyelination (Chang et

al., 2002) and astrocytic gliosis. The structural features of classical active lesions in MS have

major consequences for molecular studies. They imply that in active plaques, selected by

conventional criteria, several different molecular mechanisms take place at very close

distance, which include initial tissue injury, debris removal and repair.

Thus, target molecules identified through modern biochemical, transcriptomic or proteomic

technologies may be involved in any of these processes. Therefore, proper selection of

material, tissue micro dissection and confirmation of the results by immunocytochemistry is

essential.

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3.2.2 Microarray studies

The raw data on gene expression in different types of MS lesions have been deposited in

NCBI's Gene Expression Omnibus and are accessible through GEO Series number GSE32915

(http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE32915). As potential sources for

reactive oxygen species and the respective tissue reaction we focused on mitochondrial

genes and genes which are known to be involved in redox homeostasis, such as oxidative

burst, and in anti-oxidative defense. Highly up-or down-regulated genes in comparison to

controls were mainly seen in initial lesions and much less in established demyelinated

lesions or NAWM (Table 7).

Mitochond.

NAWM

Ox. Stress

NAWM

Mitochond.

Initial

Lesion

Ox. Stress

Initial

Lesion

Mitochond.

Early DM

Lesions

Ox. Stress

Early DM

Lesion

Down-

regulated

7 / 19 0 / 1 48/ 55 7 / 12 11 / 21 0 / 5

Up-

regulated

2/ 4 0 / 1 18/ 22 5 / 9 1 / 9 0 / 2

Table 7: Top Regulated Mitochondrial Genes and Genes Related to Oxidative Tissue Injury in Different Stages of Active MS Lesions (average values per lesion category). From the global microarray data we analysed how many genes, encoding for mitochondrial or oxidative-stress-related proteins, showed expression changes (up- or downregulated) of > 3xlog 2 fold (bold) or > 2xlog2 fold (regular)).

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3.2.3 Difference in gene expression between different cases in relation to lesion activity

All three cases included in this microarray analysis fulfilled the criteria of highly active acute

multiple sclerosis and care was taken to select comparable lesion stages from the material

by tissue micro-dissection.

Figure 12: Acute multiple sclerosis lesions used for gene expression analysis: The structure of the lesions is shown in sections stained with Luxol fast blue (myelin; a, c, e); the lower panel of figures shows the same lesions, stained for p22phox expression in activated macrophages and microglia. In the first patient (a, b) the active lesion (blue circle) is surrounded by a broad area of microglia activation with p22phox expression and myelin pallor (red circle; intial lesion), which makes it difficult to see the lesion margin in the staining for macrophages and microglia. In the normal appearing white matter (yellow circle) myelin density is normal, but there is still moderate microglia activation. ; In the second patient the demyelinated lesion core (black circle) shows concentric rings of preserved myelin. This is surrounded by the initial lesion area with extensive immunoreactivity for p22phox (red circle). The normal appearing white matter shows normal myelin density and only low expression of macrophage antigens (yellow circle). In the third patient a dense infiltrate of macrophages with p22phox expression is seen in the area of demyelination (active plaque). In the surrounding white matter there is very little expression of macrophage/microglia antigens. Areas of normal white matter used for gene expression analysis are shown by the yellow circles. Gene expression for proteins involved in oxidative damage and for mitochondrial proteins has been analysed separately in the indicated lesion areas. Dis. Dur.: disease duration.

Despite these precautions quite profound differences in gene expression were seen between

the cases. The most profound changes in gene expression were seen in case 270, a patient

with fulminant MS and disease duration of 2 weeks only. Intermediate changes were present

in case 144, who died within 4 months after disease onset and presented with a rapidly

enlarging white matter lesion with concentric demyelination. Both of these cases showed

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40

besides demyelinated lesions with massive macrophage infiltration rather large areas of

initial “pre-phagocytic” lesion areas (Fig. 12). Only moderate changes in gene expression

were seen in case 403, who died with acute MS with a clinical duration of 1.5 months. The

respective section analyzed in our study contained large demyelinated plaques with densely

packed macrophages with early myelin debris, but showed only a very small rim of initial

“pre-phagocytic” lesion area around the plaque. These data suggest a very tight regulation of

the expression of molecules involved in oxidative stress, closely depending upon the state of

activity of the lesion.

3.2.4 Mitochondrial genes

Mitochondrial genes were highly enriched in the cohort of top regulated genes (> 3 fold;

log2) in MS lesions and the most pronounced changes were seen in initial lesion areas.

Down-regulated expression was seen for 48 genes and up-regulated for 18 genes (Table 7).

All mitochondria-encoded genes, which were included in the arrays (ND1, ND2, ND3, ND5,

ND6, COX1, CYTB), were downregulated in initial MS lesions (Table 8, Fig. 13).

A similar pattern was seen for genes of the respiratory chain, with marked down-regulation,

especially of genes coding for Complex I, and Complex IV (Table 8, Fig. 13).

Downregulated Upregulated

Respiratory Chain Genes Complex I

ND1, ND2, ND3, ND5, ND6,

NDUFA3, NDUFA4, NDUFA8,

NDUFB2, NDUFB8, NDUFS5,

Complex III

CYTB,

UQCRQ

Complex IV

COX1,

COX6A1, COX6B1, COX7A2

Complex I

NDUFB10

SURF1

Table 8: Gene Expression of molecules encoding for proteins of the respiratory chain, which were up- or downregulated (> 3 log2 fold) in initial MS lesions in comparison to controls. Mitochondrially encoded genes are shown in bold; gene abbreviations and function can be seen in: www.ihop-net.org and www.sigmaaldrich.com/customer-services/services/basic-research.htlm

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Figure 13: Comparison of changes in expression level of mitochondrial respriratory chain genes between normal appearing white matter, initial lesion and early demyelinated lesion: Most profound expression changes can be observed in the initial damage zone, mainly affecting Complex I and IV. Molecules which showed changes in their gene expression higher than 2xlog2 fold compared to control are marked in blue (upregulation) or yellow (downregulation). Molecules marked in grey could not be detected with the microarrays. Non-coloured genes do not show significant alteration in their expression level.

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Regarding other mitochondrial genes with expression changes of > 3 fold (log2) again down-

regulation was seen in the majority (31), while only 16 showed up-regulated expression

(Table 9). The latter include genes involved in mitochondrial protein synthesis (MRPL18, 14,

23; MRPS15, 22), adenine nucleotide translocation (SLC25A4), and genes which are induced

by oxidative stress and are also involved in oxidative stress defense (UCP3, GRPEL1, TXNRD2,

ISCU, AASS, ACADL, DMGDH, ACADS).

Downregulated Upregulated

Other mitochondrial genes ACADVL, MRPS24, PTRH2,

FXN, ACSM2B, SLC25A17,

MRPL16, BCL2L1, PRDX3,

ALDH18A1, FDXR, CPT1B,

s75896, AW46717, APEX2,

CLPP, CYP11A1, MRPL28,

HTRA2, TUFM, FXC1,

ENDOG, MRPS18B, ARG2,

CASQ1, AF086790, NT5M,

ALDH4A1, GFM2, s81524,

MRPS25

UCP3, GRPEL1, AASS,

MRPL18, ACADL, LOC28521,

MRPS15, MRPL23, SLC25A4,

TXNRD2, ISCU, MRPS22, CS,

DMGDH, MRPL14, ACADS

Table 9: Gene expression of molecules encoding for mitochondrial proteins, which were up- or downregulated (> 3 log2 fold) in initial MS lesions in comparison to controls. Gene abbreviations and function can be seen in: www.ihop-net.org and www.sigmaaldrich.com/customer-services/services/basic-research.htlm

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3.2.5 Genes involved in radical production and response to oxidative stress

Similar as seen for the above described mitochondrial genes most pronounced changes in

the expression of genes involved in the production of reactive oxygen and nitrogen species

were seen in initial lesions, followed by demyelinated lesion areas and NAWM (Table 7). The

most pronounced changes were found for inducible (NOS2A) and endothelial (NOS3) nitric

oxide synthases and for subunits of the NADPH oxidase complex 2 (CYBA, CYBB, NCF1; Table

10). In addition, we also found enhanced expression of the ROS-generating enzymes

myeloperoxidase (MPO), eosinophil peroxidase (EPX) and lactoperoxidase (LPO), but not for

xanthine oxidase (XDH). While the expression of genes involved in the production of reactive

oxygen species were highly upregulated, that of NOS genes was reduced compared to

controls. In addition to genes involved in the production of reactive oxygen and nitrogen

species we found profound changes in the expression of genes involved in free radical

detoxification, including glutathione peroxidases, peroxiredoxins (Table 10).

Up-regulated Down-regulated

ROS Production CYBA, CYBB, NCF1

MPO, EPX, PTGS1, PXDN

NOS1, NOS2A, NOS3, NOX5, ,

MIOX, RAC1, RAC3,

ROS detoxification GPX4, PRDX1, 2, 4 GPX3, GPX5, PRDX3

Induced by ROS ALOX12, ATOX1, EPHX2,

GPR156, MSRA, STK25,

OSGIN, GLRX2, PRG3, SEPP1,

SGK2, TXNRD2

APOE; CYGB, PNKP, SCARA3,

SFTPD, SIRT2, SRXN1

Table 10: This table lists those genes, which are involved in production or detoxification of reactive oxygen species or are induced by oxidative stress and are up- or downregulated (> 2xlog2 fold changed expression values in at least 1 of 3 patients in initial MS lesions. Gene abbreviations and function can be seen in: www.ihop-net.org and www.sigmaaldrich.com/customer-services/services/basic-research.htlm

These findings further support the concept of oxidative stress as a major pathogenic factor in

initial MS lesions (Lassmann and van Horssen, 2011).

http://www.sigmaaldrich.com/customer-services/services/basic-research.htlm

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3.2.6 Immunocytochemistry for oxidative burst molecules in MS lesions:

Expression of mitochondrial proteins in different types and stages of MS lesions has been

extensively described before (Dutta et al., 2006; Lu et al., 2000; Mahad et al., 2008a; Mahad

et al., 2008b; Mahad et al., 2009; Witte et al., 2009; Witte et al., 2010) and these data were

in part obtained in sections from the same blocks and patients as those used for microarray

analysis in this study (Mahad et al., 2008b). In addition, we and others have shown before

expression of iNOS and myeloperoxidase in active MS lesions (Cross et al., 1998; Gray et al.,

2008a; Gray et al., 2008b; Liu et al., 2001; Marik et al., 2007; Zeis et al., 2009). So far,

however, data on the protein expression of molecules involved in oxidative burst in MS

lesions are not available. We, therefore next analyzed the expression of several components

of the NADPH oxidase (NOX) complexes (Fig. 14).

Nox2 complex:

The Nox2 complex is composed of two transmembrane proteins, p22phox (reflected by CYBB

in the gene expression arrays) and gp91phox (CYBA) and requires for functional activation

the association with p47phox (NCF1) as a regulatory subunit, together with p67phox (NCF2)

and p40phox (NCF4; (Bedard and Krause, 2007)). We have therefore analyzed the expression

of p22phox and gp91phox as well as p47phox as a representative of the regulatory elements

in lesions and NAWM of MS patients (Fig.12 and 14) and age-matched controls (15 a-c). The

proteins p22phox and gp91phox showed very similar expression patterns in both MS

patients and age-matched controls. In general, both proteins are expressed in all microglia

cells and macrophages (Table 11), revealing a staining pattern which is closely similar to that

seen with the pan-microglia marker Iba-1. In the white matter of controls (Fig.15 a,b) and in

the NAWM of MS patients (Fig. 14 d,e) a moderate density of p22phox and gp91phox

positive microglia was seen and these molecules were strongly expressed in microglia

nodules, when present in MS brains (Fig. 14d, e). At the lesion edge, in particular in areas of

initial (“pre-phagocytic”) lesions the expression of p22 and gp91phox was profound, due to

the marked increase in microglia density as well as to increased expression of these

molecules in individual cells (Fig. 14 dd, ee). In the demyelinated regions macrophages,

which had taken up myelin debris, expressed p22phox and gp91phox albeit to a lesser extent

than in the initial lesion area (Fig. 14 c, ddd, eee). In contrast, the expression pattern of

p47phox (NCF1) was much more restricted. In the white matter of controls only few

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45

perivascular macrophages were stained (Fig.15 c). In MS lesions we found it in only 5 to 20%

of macrophages and activated microglia and this was predominantly localized in areas of

initial tissue damage at the edge of actively demyelinating lesions (“pre-phagocytic” lesion

areas; Fig. 14 f-fff).

Figure 14 a-c: Active lesion in a patient with primary progressive multiple sclerosis;

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Figure 14 a-c: Active lesion in a patient with primary progressive multiple sclerosis; a: luxol fast blue (LFB) myelin staining shows a demyelinated lesion with defined borders; b: In the adjacent section stained for p22phox profound expression is seen at the active lesion edge, spanning into the adjacent normal appearing white matter (initial lesion area); c: In the inactive centre of the lesion p22phox is weakly expressed in some macrophages. d-hhh: These images show the expression of oxidative burst-associated molecules in the normal appearing white matter (left column), in the zone of initial tissue injury (middle column) and in the demyelinated zone (right column). Most pronounced expression of all proteins is seen in the initial lesion area (middle panel), while expression for p22phox and gp91phox is much weaker in lipid containing macrophages in the lesion center (right panel). In the normal appearing white matter microglia nodules can be seen, which are intensely stained for p22phox, gp91phox and NOX1. P22phox, gp91phox and p47phox are only expressed in macrophages and microglia, while Nox1 shows a broader expression also in astrocytes (asterisk in gg) and endothelial cells (asterisk in ggg labels the vessel with endothelial staining); the expression of Noxo1 is even broader compared to that of Nox1. P47phox staining is absent in the normal appearing white matter (f), while profound expression is seen in macrophages and small microglia like cells in the initial damage zone (ff); the insert in ff shows expression of p47phox (red) in macrophages stained with LN3 (green). In the lesion center only weak reactivity for p47phox is seen, mainly in perivascular macrophages (fff, in the insert at higher magnification). i-o: Confocal laser microscope images of double staining with different Nox markers and CNS cell specific markers; the staining combinations are indicated on the figures; these data show co-localization of different Nox components within the same macrophages or microglia cells in the MS lesions. Nox 1 is also expressed in GFAP positive astrocytes (n) in the absence of p22phox (o). Red and green staining depict the individual antigens as indicated in the figure; yellow staining represents double staining.

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Figure 15: a to e show the expression of different NOX markers in the normal white matter of a control patient. P22phox (a) and gp91phox (b) are expressed in moderate intensity in microglia. P47phox staining (c) is absent in the brain parenchyma, but is seen in some perivascular macrophages. (d) NOX1 shows very weak expression in some microglia, astrocytes and endothelial cells, while NOXO 1 (e) is only weakly expressed in some microglia-like cells. The magnification bar represents 50µmand applies for pictures a-j. Figs. f-i show double staining for Class 1 restricted T-cells (CD8; black) and p22phox expression in macrophages/microglia. P22phox expression in phagocytes is associated with moderate infiltration of the lesions by CD8+ T-lymphocytes. Fig.f shows a microglia nodule in the NAWM associated with a single CD8+ T-cell. Fig. g shows the transition area between the NAWM and the initial lesion zone with some clusters of T-cells in the parenchyma and much higher density of p22phox expressing cells. Fig. h was taken from the initial lesion area with some diffuse T-cell infiltration and profound p22phox expression. In Fig. i the demyelinated center of the active lesion is shown with lower expression of p22phox in macrophages and some perivascular and parenchymal T-cells. Fig. j: Double staining for CD20 positive B-cells (blue) and p22phox (brown). Most profound p22phox expression is seen in macrophages and microglia. Some of the B-cells show weak brown staining, suggesting a moderate expression of p22phox.. Fig. k: Confocal laser microscopic double staining for CD3 (pan T-cells; green) and p22phox (red) in a thick perivascular inflammatory infiltrate. No co-localization is seen. Fig. l: Absence of double staining by CD8 (green) and p22phox (red) in the inflammatory infiltrates. Fig. m: As suggested by picture j expression of p22phox (red) is seen in CD20+ B-lymphocytes (green/yellow). The magnification bar in k-m represents 50µm.

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48

The quantitative analysis of p22phox expression by densitometry revealed additional insights

(Fig. 16 a). In classical active lesions from early MS the expression in the lesions was very

high, but in the surrounding NAWM it was similar to that seen in normal white matter of

controls. In contrast, in slowly expanding lesions p22phox expression in the active lesion

parts was, as expected, lower than in classical active lesions and this apparently reflects the

lower/milder degree of active tissue injury. Finally, p22phox expression was very low in the

center of inactive lesions, reflecting reduced microglia density in these areas in comparison

to normal white matter (Lassmann, 2011). A very similar expression pattern was seen for the

gp91phox antibody resulting in a significant correlation between p22phox and gp91phox

expression (Fig.16 b).

The expression of p22phox, seen in different types of MS lesions, in general co-localized in

the same areas with the presence of oxidized DNA (Fig. 16 c) and lipids, described in detail

earlier (Haider et al., 2011). In addition, we found a significant correlation between the

extent of p22phox expression with the number of dystrophic axons, immunoreactive for

amyloid precursor protein (R=0.47; p<0.001) and oxidized phospholipids (R=0.35; p<0.006;

Fig. 16 d), with the number of CD3+ T-cells (R=0.55; p<0.001) and the number of HLA-DR+

microglia cells and macrophages (R=0.69; p<0.001). The values for inflammatory cells,

dystrophic axons and for oxidized DNA and lipids have been determined in previous studies

on the same material as used in the present study (Frischer et al 2009, Haider et al 2011).

We did not find significant differences of p22phox expression with regard to gender. There

was, however, a significant decrease of p22phox expression with disease duration (R=0.18;

p<0.021). Patients with acute or RRMS showed significantly more lesional p22phox

expression than patients who died during the progressive stage of the disease (PPMS and

SPMS; p<0.012).

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Figure 16 a: Quantitative analysis of p22phox expression in different types of multiple sclerosis lesions. In comparison to white matter of controls there is a significantly higher expression (p < 0.01) in active and slowly expanding MS lesions, and in the normal appearing white matter of slowly expanding lesions; in the lesions p22phox positive microglia are mainly seen in the active lesion edge (initial lesions) and less in the inactive lesion center. Furthermore, we found a significant decrease of p22 expressing microglia in the center of inactive lesions. b: Correlation between p22 and gp91 expression in different MS cases and lesions. The same areas of NAWM and lesions were scanned for p22phox and gp91phox expression and regression was analysed as described in materials and methods. c: Comparison between p22phox expression, determined by densitometry and the number of nuclei with oxidized DNA (8OHdG immunoreactivity) within MS lesions; d: Comparison between p22phox expression and the number of dystrophic axons, immunoreactive for oxidised phospholipids (E06); p22phox and gp91phox immunoreactivity was determined by densitometry; nuclei with oxidised DNA and dystrophic axons, positive for E06 were counted manually (Haider et al., 2011).

A more detailed analysis of p22phox expression in relation to inflammation showed the

presence of T-cells (mainly CD8+ cells) associated with profound p22phox expression in

microglia and macrophages (Fig. 15 f-i). This was even seen in microglia nodules in the

NAWM (Fig. 15 f). In double staining we did not find co-localization of p22phox with T-cell

markers (CD3, CD8; Fig. 15 k, l), however, p22phox was apparently expressed in a subset of

CD20+ B-lymphocytes (Fig. 15 j, m).

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50

Nox1 complex:

The Nox1 complex contains two trans-membrane proteins (p22phox and Nox1), as well as

cytoplasmic regulatory molecules (Noxo1 and Noxa1; (Bedard and Krause, 2007; Cheret et

al., 2008). The expression patterns of Nox1 and Noxo1 were different from those of the

Nox2 complex (Table 11). In 9 out of 16 controls Nox1 was weakly expressed in some

microglia, astrocytes and endothelial cells (Fig. 15 d) while no staining was seen in the

others. Very weak Noxo1 expression was exceptionally detected in microglia in controls

(Fig15 e). In MS lesions Nox1 and Noxo1 expression was mainly seen in and around active

plaques of acute and relapsing MS (Fig. 14 g, gg, ggg and h, hh, hhh). Nox1 was not only

present in macrophages and microglia, but also in astrocytes and endothelial cells (Fig. 14 gg,

ggg asterisks). Expression was not restricted to initial (“pre-phagocytic”) areas but more

generally throughout the plaque area and the adjacent NAWM, where it was found in

microglia nodules (Fig. 14 g). In slowly expanding lesions some Nox1 staining was found in

microglia, macrophages, astrocytes and endothelial cells at the active lesion edge and in the

adjacent NAWM. Overall, Noxo1 immunoreactivity was very weak and mainly present in

initial lesions. Its expression was more diffuse, but accentuated in microglia and

macrophages. Immunoreactivity in inactive lesions was similar to that seen in controls.

The co-localization of the different Nox subunits in the same cell-type in MS lesions suggests

functionally active oxidative burst:

Superoxide generation through Nox molecules requires functionally assembled subunits.

We, thus, performed double labeling with confocal laser microscopy to test for co-

localization of the respective molecules in the same cells (Fig.14 i-o and 17, Table 11). In

macrophages and microglia we identified co-localization of all the tested components of the

Nox1 and Nox2 complexes. This was, however, not the case for Nox1 complex expression in

astrocytes and endothelial cells. In these cells no convincing expression of p22phox was seen

(Fig. 14 n, o and 17).

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51

Table 11: Double stainings were performed with markers for NADPH subunits and for microglia (Iba1 and CD68) or astrocytes (GFAP). Stained sections were analyzed by confocal laser microscopy as shown in Fig. 14 and 17. “n.p”. stands for “staining not possible” (primary polyclonal antibodies derived from same species).

Figure17: Cellular localization of NADPH-Oxidase subunits: Expression of NADPH- components on astrocytes (GFAP) and microglia/macrophages (CD68, LN3) was tested. (+) indicates colocalization on the same cell-type whereas (-) indicates no colocalization.

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52

Follow-up studies on primary rat glia cultures showed that p22phox expression could be

detected on cells with microglia-like morphology but not on astrocytes (results consistent

over 3 repetitions from duplicates). Stimulation with TNFα, mimicking inflammatory

conditions, did not lead to expression of p22phox on GFAP positive astrocytes either (Fig.

18).

Figure 18: Absence of p22phox expression on rat primary astrocytes in cell culture. Double labeling with the NADHP-Oxidase subunit p22phox and a marker for astrocytes (GFAP) shows no colocalization in untreated or TNFα treated cells.

Discussion

53

4 Discussion

(Fischer et al, 2011)

A major prerequisite for gene expression studies is that the pathological material used is

exactly defined and characterized. In multiple sclerosis this is difficult, since several types of

active lesions exist, depending upon the stage of the disease, the age and location of these

lesions, and the inter-individual differences between patients. In addition, within an active

lesion different closely adjacent zones are present reflecting initial tissue injury, debris

removal or repair. Here evidence is reviewed, showing that distinct subareas of active MS

lesions reflect different pathological hallmarks of lesion evolution. These data provide the

basis for our understanding of the pathogenesis of tissue injury in MS and imply that studies

on MS pathogenesis have to rely on a clear definition of the lesions analyzed and have to

focus on specific lesion areas, isolated by micro dissection.

Our current results on the expression pattern of subunits of the NADPH oxidase complex

expand previous observations, which suggest a prominent role of oxidative injury in the

pathogenesis of demyelination and tissue injury in MS (Bagasra et al., 1995; Bizzozero et al.,

2005; Smith et al., 1999; Smith and Lassmann, 2002; van Horssen et al., 2011; Vladimirova et

al., 1998). In a recent study we found that oxidized DNA and lipids are present in high

amounts in active MS lesions, in particular at sites of initial tissue injury. Furthermore, the

presence of oxidized epitopes was enriched in apoptotic oligodendrocytes and in acutely

injured dystrophic axons (Haider et al., 2011). The prominent up-regulation of gene

expression of molecules, induced by oxidative stress/ or involved in redox homeostasis, as

seen in our current study provides additional support for the contribution of reactive oxygen

species (ROS) in the pathogenesis of early MS.

ROS production is accomplished by two principally different mechanisms; activation of free

radical-producing enzymes, such as those involved in oxidative burst, and by mitochondrial

dysfunction (Smith, 2011; van Horssen et al., 2010; Witte et al., 2010). Support for both

mechanisms comes from our microarray study since we found profound changes in the

expression of mitochondrial genes and, in particular of those encoded by mitochondrial

DNA. The present results are in line with previous biochemical, histochemical and

Discussion

54

immunocytochemical studies, which showed a profound impairment of mitochondrial

function in active MS lesions, which appears to be related to active degeneration of myelin,

oligodendrocytes, axons and neurons (Mahad et al., 2008b). Mitochondrial dysfunction is

transient, as seen in the comparison of initial with demyelinated lesion areas in our arrays.

At later stages of lesion formation mitochondrial numbers and enzyme activity increase,

apparently reflecting the increased metabolic demand of demyelinated axons in the lesions

or a reaction to chronic mitochondrial insult (Mahad et al., 2009; Witte et al., 2009). Taken

together, it is therefore likely that dysfunction of the mitochondria contributes to ROS

production within MS lesions. It is, however, unlikely that this phenomenon is responsible

for the initial step of mitochondrial dysfunction in the lesions.

Our microarray data, in combination with the immunohistochemical results, identify

activated macrophages and microglia as the major source for ROS production in initial MS

lesions. We demonstrated increased expression of the major components of the Nox2 and

Nox1 complexes in active MS lesions, predominantly in areas of initial “pre-phagocytic”

tissue injury, as defined by Barnett (Barnett and Prineas, 2004), Marik (Marik et al., 2007)and

Henderson et al (Henderson et al., 2009a). This is the area of active MS lesions, where

myelin sheaths are still preserved, but distal oligodendrogliopathy, oligodendrocyte

apoptosis and acute axonal injury take place in association with mild T cell infiltrates and

profound microglia activation (Barnett and Prineas, 2004; Lassmann, 2011; Lucchinetti et al.,

2000; Marik et al., 2007). Furthermore, cells containing oxidized DNA and oxidized lipids are

mainly concentrated at these sites (Haider et al., 2011) and the most pronounced damage to

mitochondria in oligodendrocytes and axons is seen in this area (Mahad et al., 2008b). In

fact, many of these observations have been made in exactly the same tissue blocks from the

same patients, as described in the present study. Experimental studies suggest that oxidative

tissue damage under these conditions is most likely mediated by peroxynitrite. Nitrotyrosine

expression, a footprint of peroxynitrite-induced injury, has been found at the edge of active

MS lesions (Zeis et al., 2009) and is known to mediate oligodendrocyte injury in vitro and in

autoimmune encephalomyelitis in vivo (Li, 2005; Nikic et al., 2011; Vana et al., 2011). Our

present data strongly suggest that ROS, which are also necessary for peroxynitrite formation,

are mainly produced by activated microglia through classical Nox2-dependent oxidative

burst. This view is supported by several observations. First, p22phox and gp91phox are much

more abundantly expressed in active MS lesions as compared to other oxidases, such as for

Discussion

55

instance myeloperoxidase (see arrays and Marik and Gray (Gray et al., 2008a; Gray et al.,

2008b; Marik et al., 2007). Secondly, the co-expression of different components of the Nox2

complex in the same microglia cells indicates that these complexes are functionally active.

Thirdly, it is interesting to note that p22phox and gp91phox expression is less intense in

macrophages that have taken up myelin debris in comparison to microglia in the initial lesion

zone. This observation supports the concept that myelin phagocytosis de-activates

macrophages from a pro-inflammatory to an anti-inflammatory phenotype within MS

lesions. Potential functional importance of Nox2 complexes in inflammatory demyelinating

brain lesions is shown by the protective effect of gp91phox gene deletion in animals with

autoimmune encephalomyelitis (Li et al., 2011). Furthermore, Nox2 attenuation, by either

genetic knockdown or pharmacological compounds, is beneficial in animal models for

neurodegeneration. Nox2 deficiency reduced oxidative stress and improved the outcome in

a mouse model of Alzheimer’s disease (Park et al., 2008) and neurodegeneration was

markedly attenuated in an experimental animal model of Parkinson’s disease compared with

wild-type animals (Zhang et al., 2004).

In contrast, little is known about the role of the Nox1 complex in MS and experimental brain

inflammation. In vitro, microglia toxicity is in part mediated through ROS production by the

Nox1 complex (Cheret et al., 2008). In addition, Nox1 expression was not restricted to

macrophages and microglia, where p22phox is present for potential interaction. In

astrocytes and endothelial cells Nox1 and Noxo1 were present in the absence of detectable

p22phox. Whether astrocytic and endothelial Nox1 is also able to produce ROS in the

absence of p22phox or whether they serve other functions in these cells warrants future

studies.

Despite the use of formaldehyde fixed paraffin embedded material we found a very good

correspondence between the gene expression data obtained in microarrays and the

respective immunohistochemical results. This was not only the case for the changes in

relation to oxidative burst molecules, which were directly analyzed here, but also for those

related to mitochondrial function, where respective immunocytochemical analysis has been

performed on the same material before (Mahad et al., 2008b). It has been shown earlier that

transcriptome analysis can be done on paraffin embedded material (Lewis et al., 2001; von

Weizsacker et al., 1991; Waddell et al., 2010) and we show that this is even feasible on

archival autopsy material from MS patients. This is important, since acute MS lesions are

Discussion

56

very rare in pathological collection and so far not available in native frozen tissue blocks.

There were, however a number of exceptions and caveats. First, some mRNAs, such as for

instance that for iNOS, were down-regulated in the arrays, despite profoundly increased

protein expression seen in immunocytochemistry (Marik et al., 2007). In the normal white

matter of the human brain a low number of iNOS positive microglia cells is present (Marik et

al., 2007), which may reflect some basic activation of microglia in the human brain in

comparison to animals, housed under specific pathogen free conditions. This may explain

the moderate basic level of iNOS mRNA expression in controls seen in our microarrays. Since

iNOS mRNA expression after cytokine stimulation is transient due to its instability and to

active regulation by the presence of nitric oxide (Murphy, 2000; Park et al., 1997), down-

regulation in the active MS lesions may not be unexpected. Secondly, one third of the 33.000

genes showed very low expression levels, which also showed no changes between cases or

between MS patients and controls. These values had to be excluded from the analysis due to

insufficient RNA quantity. This is apparently a technical limit of gene expression studies in

archival, formaldehyde fixed and paraffin embedded tissue material, having a much lower

sensitivity compared to those performed on native frozen tissue (Waddell et al., 2010).

Finally, more close inspection of the data showed profound differences between the cases.

As discussed above these differences may best be explained by the complexity of lesion

architecture and more subtle differences in the stage of the respective lesions (Lassmann,

2011). These data suggest that in a disease, such as MS, transcriptome or proteome analysis

should not be performed by simply comparing active with inactive lesions or control white

matter, using standard criteria for lesion definition. What is needed for this type of research

in the future is a highly precise area selection and micro-dissection, similar but possibly even

better than that done in our current study.

One can argue that the brain samples, selected for gene expression analysis in our study are

not derived from typical MS cases. In fact, all three were from patients with acute MS and

one showed prominent concentric demyelination, typical for Balo’s disease. There are,

however, two main arguments, which support our view, that the changes seen in these cases

are representative for more classical MS lesions. First, the same changes, although in lower

severity, were seen by immunocytochemistry in all other active MS lesions, including even

the slowly expanding lesions of progressive MS. Secondly, it has been shown before that

those tissue alterations, characteristic for pattern III lesions, including elements of concentric

Discussion

57

sclerosis, are seen to a lesser extent in other classical active lesions from patients with

relapsing or progressive MS (Barnett and Prineas, 2004; Marik et al., 2007).

In conclusion, our data provide further evidence for the importance of oxidative damage in

the pathogenesis of demyelination and tissue injury in MS. We suggest that tissue damage is

initiated by oxidative burst in activated microglia and macrophages, which is most likely

induced by the inflammatory process. Oxidative damage leads to mitochondrial injury and

disturbance of the mitochondrial respiratory chain, which not only results in energy

deficiency but also in further propagation of ROS production (Lassmann and van Horssen,

2011; Smith, 2011). Neuroprotective therapies, specifically focusing on the prevention of

oxidative damage may, thus, become attractive in the future and a current trial, testing the

effect of fumarates, which boost endogenous antioxidant enzymes, in MS patients,

represents one possible example for this approach (de Vries et al., 2008; Linker et al., 2011;

Schreibelt et al., 2007). However, experimental data have shown that RNS as well as ROS

can, under certain circumstances, also mediate beneficial, anti-inflammatory effects in

autoimmune encephalomyelitis (Becanovic et al., 2006; Liu et al., 2006; Sahrbacher et al.,

1998; Willenborg et al., 2007), possibly by repressing the T-cell mediated immune response.

On this basis stimulation of ROS production has been suggested as a potential therapy for

MS patients (Becanovic et al., 2006). In light of our current observations such therapeutic

trials should be met with caution.

58

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Acknowledgements

I would like to thank all the people that helped me in all various concerns during the

last years of working on my PhD project:

First of all, I want to thank my supervisor Prof. Hans Lassmann for the opportunity to

work and study in his lab, for his persistent support over the past years, especially

for always finding time for discussions, for giving me the possibility to constantly

improve my performances by encouraging me to give challenging talks, but also for

sending me to various conferences thereby providing newest insights into the

complex matter of Multiple Sclerosis as well as chances for further education and

networking. His support and his trust in me contributed considerably to the

development of my scientific and personal self-confidence.

I also want to thank Doz. Monika Bradl for initially arousing my interest for the field

of neuroimmunology and her support during the whole time that I spent at the

Center for Brain Research.

Thanks to my whole family for loving and supporting me all my life without any

constraints (although or especially because –retrospectively- it wasn’t always easy

for them).

I also want to thank my working colleagues and -friends for their constant scientific

and social assistance and help. Here, I want to mention Eva Maria Nicolussi, Rakhi

Sharma and Lukas Haider for their close cooperation but also Maila Klügling, Roland

Prielhofer, Rui Martins, Cornelia Schuh, Isabella Wimmer, Maja Kitic, Simon

Hametner, Susanne Laukoter, Tim Stuhlmann and Milena Adzemovic.

I also want to thank my CCHD colleagues for the time we shared together.

Special thanks to Ulli Köck, Marianne Leisser and Angela Kury for the excellent

technical assistance as well as for help and discussions that facilitated my work a lot.

Thanks also to Jamie Lim and Jack van Horssen from the Amsterdam group, it was a

pleasure to cooperate with them for this project.

66

And last but not least I want to thank some of my close friends for their company

and loyalty:

Milos, Zdravko, Flo, Marco, Eva and Maila for being not only colleagues but

becoming an important part of my life that I don’t want to miss in the future.

Babsi, Georg, Mitzka, Stefan and Patrick for their longtime-friendship, their support

when I’m down and for their company whenever I needed distraction.

Renate and Karl for saving me when despair takes over, for always providing shelter,

allowing me to recharge my batteries and enabeling me to regain the energy that I

need to accomplish my work and my life.

And finally once more my sisters Julia and Caro, for being my everything.

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List of publications

NADPH Oxidase Expression in Active Multiple Sclerosis Lesions in Relation to Oxidative Tissue Damage and Mitochondrial Injury. Fischer MT, Sharma R, Lim J, Haider L, Frischer J, Mahad D, Bradl M, Van Horssen J, Lassmann H (Brain, under revision)

Oxidative damage in multiple sclerosis lesions . Haider L, Fischer MT, Frischer JM, Bauer J, Höftberger R, Botond G, Esterbauer H, Binder CJ, Witztum JL, Lassmann H. (Brain, 2011, 134(Pt 7): 1914-1924)

Pathogenic T cell responses against aquaporin 4. Pohl M, Fischer MT, Mader S, Schanda K, Kitic M, Sharma R, Wimmer I, Misu T, Fujihara K, Reindl M, Lassmann H, Bradl M. (Acta Neuropathol, 2011, Apr 6. [Epub ahead of print])

Inflammation induced by innate immunity in the central nervous system leads to primary astrocyte dysfunction followed by demyelination. Sharma R, Fischer MT, Bauer J, Felts PA, Smith KJ, Misu T, Fujihara K, Bradl M, Lassmann H. (Acta Neuropathol, 2010, 120(2): 223-236)

The 'window of susceptibility' for inflammation in the immature central nervous system is characterized by a leaky blood-brain barrier and the local expression of inflammatory chemokines. Schoderboeck L, Adzemovic M, Nicolussi EM, Crupinschi C, Hochmeister S, Fischer MT, Lassmann H, Bradl M. (Neurobiol Dis, 2009, 35(3): 368-75)

After injection into the striatum, in vitro-differentiated microglia- and bone marrow-derived dendritic cells can leave the central nervous system via the blood stream. Hochmeister S, Zeitelhofer M, Bauer J, Nicolussi EM, Fischer MT, Heinke B, Selzer E, Lassmann H, Bradl M (Am.J.Pathol., 2008, 173(6): 1669-1681)

68

CURRICULUM VITAE

Döblinger Hauptstraße 18

A-1190 Wien

+43 699 11085030

[email protected]

[email protected]

Maga. Marie-Therese Fischer

Personal Data

Date of birth 12.May 1984

Place of birth Vienna

Nationality Austria

Marital status single

Education

09/2007-09/2011 PhD Studies, Medical University of Vienna

CCHD Program (Cell Communication in Health and Disease)

Center for Brain Research, Div. of Neuroimmunology/

Head: Univ. Prof. Dr. Hans Lassmann Supervisor: Univ. Prof. Dr. Hans Lassmann

„Cortical Demyelination in Progressive Multiple Sclerosis“

10/2002-10/2007 Graduate Studies in Molecular Biology, emphases:

Neurobiology, Immunology, Genetics

Faculty of Life Science, University of Vienna

Master thesis at the Medical University of Vienna

Center for Brain Research, Div. of Neuroimmunology/

Head: Univ. Prof. Dr. Jürgen Sandkühler Supervisor: Univ. Doz. Dr. Monika Bradl

„Plasticity of microglia cell activation”

1994-2002 Public secondary school, emphases: languages

BG18,A-1180 Klostergasse 25

mailto:[email protected]

mailto:[email protected]

69

Scientific presentations

2009-2011 Annual PhD-Symposium of the Young Scientist

Association (YSA)

2009-2011 Annual Meeting of the Max-Planck Society, Division of Neuroimmunology, Ringberg, Germany

„From gene expression studies to disease mechanisms“ (2011)

„Cortical demyelination in progressive Multiple Sclerosis “ (2010)

„Molecular triggers for demyelination in progressive Multiple Sclerosis” (2009)

Various presentations at the Center for Brain Research

„Work in progress“

Science communication presentations

2011 Brain Awareness Week, Center for Brain Research

„Kampf der Giganten – Immunsystem versus Zentralnervensystem“

Workshop organization

2010 Bridging the Gap II

2009 Bridging the Gap I

Links

http://www.meduniwien.ac.at/phd-cchd/

http://cbr.meduniwien.ac.at/

http://www.meduniwien.ac.at/phd-cchd/workshop/

Maga.Marie-Therese Fischer Vienna, November 2011

http://www.meduniwien.ac.at/phd-cchd/

http://cbr.meduniwien.ac.at/

http://www.meduniwien.ac.at/phd-cchd/workshop/

Documents

NADPH-Oxidase in Active Multiple Sclerosis Lesions · 1.1 Multiple Sclerosis and its pathology Multiple Sclerosis (MS) is the most common neurological disease in young adults in the