University of Veterinary Medicine Hannover

Institute for Animal Ecology and Cell Biology

Division of Cell Biology

Nitric oxide (NO)- and carbon monoxide (CO)-

mediated signal transduction in a co-culture system

of microglia and human model neurons

THESIS

submitted in partial fulfillment of the requirements for the degree

- DOCTOR RERUM NATURALIUM -

(DR. RER. NAT.)

by

Hannah Christina Scheiblich, M.Sc.

Cologne, Germany

Hannover 2015

Supervisor: Prof. Dr. Gerd Bicker

Division of Cell Biology, Institute for Animal Ecology and Cell Biology, University of Veterinary Medicine Hannover, Germany

Supervision group: Prof. Dr. Gerd Bicker

PD Dr. Michael Stern Division of Cell Biology, Institute for Animal Ecology and Cell Biology, University of Veterinary Medicine Hannover, Germany

1st Evaluation: Prof. Dr. Gerd Bicker Division of Cell Biology, Institute for Animal Ecology and Cell Biology, University of Veterinary Medicine Hannover, Germany

2nd Evaluation: Apl. Prof. Dr. Manuela Gernert Department of Pharmacology, Toxicology and Pharmacy, University of Veterinary Medicine Hannover, Germany

Date of final exam: 09.11.2015

The present work was supported by a grant from the German Research Foundation

(DFG) to Gerd Bicker (FOR 1103, BI 262/16-2).

Dedicated to

my family

Table of Contents I

Table of Contents

List of publications .............................................................................. III

Abbreviations ....................................................................................... V

Abstract ...............................................................................................VII

Zusammenfassung ..............................................................................IX

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

Gatekeepers of the CNS: Microglia in health and disease ...................................... 2

Cell-to-cell communication signaling ....................................................................... 5

Nitric oxide: the gas with a dual character ...................................................................... 6

Carbon monoxide: a novel agent to counteract inflammation? ....................................... 8

Feedback between nitric oxide and carbon monoxide generation ................................... 9

Aims of the dissertation ..................................................................... 12

Publications ......................................................................................... 13

Authors’ contributions ............................................................................................ 13

Nitric oxide / cyclic GMP regulates motility of a microglial cell line and primary

microglia ................................................................................................................ 15

Nitric oxide regulates antagonistically phagocytic and neurite outgrowth inhibiting

capacities of microglia ........................................................................................... 16

Regulation of microglial migration, phagocytosis and neurite outgrowth by HO-

1/CO signaling ....................................................................................................... 17

Enhanced neurite outgrowth of human model (NT2) neurons by small-molecule

inhibitors of Rho/ROCK signaling .......................................................................... 18

Discussion ........................................................................................... 19

BV-2 cells as model system for primary microglia ................................................. 21

Microglial activation: why could carbon monoxide be beneficial for the inflamed

brain? .................................................................................................................... 22

Reciprocity of nitric oxide and carbon monoxide in regulating microglial cell

migration ............................................................................................................... 24

Antagonistic regulation of microglial phagocytosis by nitric oxide and carbon

monoxide ............................................................................................................... 26

Neurite outgrowth of human neurons is regulated by microglia ............................. 28

II Table of Contents

Conclusion .......................................................................................... 31

References ........................................................................................... 32

Acknowledgements ............................................................................ 41

Eidesstattliche Erklärung ................................................................... 43

List of publications III

List of publications

Parts of this thesis were already published

SCHEIBLICH H, BICKER G (2015) Nitric oxide regulates antagonistically phagocytic and

neurite outgrowth inhibiting capacities of microglia. Developmental Neurobiology, accepted

for publication. DOI: 10.1002/dneu.22333

ROLOFF F, SCHEIBLICH H, DEWITZ C, DEMPEWOLF S, STERN M, BICKER G (2015) Enhanced

neurite outgrowth of human model (NT2) neurons by small-molecule inhibitors of Rho/ROCK

signaling. PLoS ONE 10(2): e0118536. DOI: 10.1371/journal.pone.0118536

SCHEIBLICH H, BICKER G (2015) Regulation of microglial migration, phagocytosis, and

neurite outgrowth by HO-1/CO signaling. Developmental Neurobiology 75(8): 854-876. DOI:

10.1002/dneu.22253

SCHEIBLICH H, ROLOFF F, SINGH V, STANGEL M, STERN M, BICKER G (2014) Nitric

oxide / cyclic GMP regulates motility of a microglial line and primary microglia. Brain

Research 1564: 9-21. DOI: 10.1016/j.brainres.2014.03.048

Other publications (not relevant for thesis)

SIJU KP, REIFENRATH A, SCHEIBLICH H, NEUPERT S, PREDEL R, HANSSON B, SCHACHTNER

J, IGNELL R (2013) Neuropeptides in the antennal lobes of the yellow fever mosquito, Aedes

aegypti. The Journal of Comparative Neurology, 522(3), 592-608. DOI: 10.1002/cne.23434

EICKHOFF R, LORBEER RA, SCHEIBLICH H, HEISTERKAMP A, MEYER H, STERN M, BICKER G

(2012) Scanning Laser Optical Tomography resolves structural plasticity during regeneration

in an insect brain. PLOS ONE, 7, e41236. DOI: 10.1371/journal.pone.0041236

STERN M, SCHEIBLICH H, EICKHOFF R, DIDWISCHUS N, BICKER G (2012) Regeneration of

olfactory afferent axons in the locust brain. The Journal of Comparative Neurology, 520(4),

679-693. DOI: 10.1002/cne.22770

IV List of publications

Conference contributions regarding this thesis

SCHEIBLICH H, BICKER G (2015) Nitric oxide-mediated microglial phagocytosis: why could

carbon monoxide be good for the inflamed brain?, XII European Meeting on Glial Cells in

Health and Disease, T12-62A, Bilbao, Spain.

SCHEIBLICH H, BICKER G (2015) Nitric oxide-mediated microglial phagocytosis and why

carbon monoxide could be good for the inflamed brain, Symposium „Advances in Research

on Neurodegenerative Disease with a Focus on Dementias“, Halle (Saale), Germany.

SCHEIBLICH H, BICKER G (2015) Anti-inflammatory role of heme oxygenase-1 / carbon

monoxide in functional assays using co-cultures of microglia and human model neurons, 11th

Göttingen Meeting of the German Neuroscience Society, T12-3A, Göttingen, Germany.

SCHEIBLICH H, ROLOFF F, SINGH V, STANGEL M, STERN M, BICKER G (2014) Nitric oxide /

cyclicGMP regulates motility of a microglial line and primary microglia, 9th FENS Forum of

Neuroscience, B021, Milan, Italy.

SCHEIBLICH H, ROLOFF F, SINGH V, STANGEL M, STERN M, BICKER G (2014) Nitric oxide /

cyclicGMP regulates motility of microglia in vitro, 2nd International Workshop of Veterinary

Neuroscience, Hannover, Germany.

SCHEIBLICH H, ROLOFF F, SINGH V, STANGEL M, STERN M, BICKER G (2013) Nitric oxide /

cyclicGMP regulates motility of a microglial cell line, 10th Göttingen Meeting of the German

Neuroscience Society, T9‐5B, Göttingen, Germany.

Abbreviations V

Abbreviations

Ca2+ calcium

cGMP cyclic guanosine monophosphate / zyklisches

Guanosinmonophosphat

CNGC cyclic nucleotide gated ion channels

CNS central nervous system

CO carbon monoxide / Kohlenmonoxid

CORM CO-releasing molecule / CO-freisetzendes Molekül

CORM-II CO-releasing molecule: Tricarbonyldichlororuthenium(II) dimer

Fe2+ ferrous iron

GMP guanosine monophosphate

GTP guanosine triphosphate

HO heme oxygenase

HO-1 heme oxygenase-1 / Hämoxigenase-1

iNOS inducible nitric oxide synthase / induzierbare NO-Synthase

LPS lipopolysaccharide

NF-ƙB nuclear factor kappa-light-chain enhancer of activated B cells

NO nitric oxide / Stickstoffmonoxid

NOS nitric oxide synthase

Nrf2 nuclear factor-erythroid 2-related factor 2

NT2 human Ntera2/D1 precursor cells

O2- superoxide

ONOO- peroxynitrite

PDE cyclic nucleotide phosphodiesterase

PHOX phagocytic oxidase

PKG protein kinase G

PRRs pattern-recognition receptors

PS phosphatidylserine

RhoA Ras homolog gene family, member A GTPase

VI Abbreviations

ROCK Rho-associated coiled coil forming protein serine/threonine

kinase

sGC soluble guanylyl cyclase / lösliche Guanylatzyklase

TLR Toll-like receptor

Xkr8 Xk-related protein 8

ZNS Zentralnervensystem

Abstract VII

Abstract

Hannah Scheiblich

Nitric oxide (NO)- and carbon monoxide (CO)-mediated signal transduction in a

co-culture system of microglia and human model neurons

Inflammation within the brain is usually accompanied by the activation of microglia

and is commonly associated with various neurodegenerative diseases. Microglia are

the resident immune effector cells of the central nervous system (CNS), initiating a

range of cellular responses for host defense upon recognizing damage or danger

signals. However, it is widely accepted that microglia play a dual role in mediating the

immune response since it has been shown that failed regulation of microglial

activation can exacerbate the progression of neurodegeneration. The aim of this

thesis was to investigate the regulation of different cellular characteristics of activated

microglia in response to the two gaseous messenger molecules nitric oxide (NO) and

carbon monoxide (CO).

In the first part of this thesis, mechanistic links between lipopolysaccharide (LPS)-

induced inflammation, NO signaling, microglial activation, and cell migration were

explored in an in vitro approach of cells from the microglial cell line BV-2 and rodent

primary microglia. The migration pattern of BV-2 cells versus primary microglia was

investigated by employing small bioactive enzyme activators and inhibitors of the

NO/cGMP signaling cascade. Despite some differences in the threshold towards

stimulation with the chemical agents, it could be demonstrated that NO positively

regulates the cell migration of microglia via cyclic guanosine monophosphate

(cGMP), thereby leading to cytoskeletal rearrangement. Moreover, the microglial cell

line BV-2 was established as an adequate model system for primary microglia.

The second part of this thesis evaluated the regulatory effects and cross-talk

between NO and CO on certain aspects of microglial biology, including activation,

migration, phagocytosis and neuron-interaction. Data from my cell culture model

show that LPS-stimulated microglia increase their NO production via activation of the

inducible NO synthase (iNOS), resulting in an increase in their phagocytotic activity

VIII Abstract

through self-stimulation by a mechanism independent of the sGC/cGMP pathway.

Stimulation of the CO-generating enzyme heme oxygenase-1 (HO-1) and application

of a CO-donor prevented the production of NO during LPS stimulation, and

attenuated microglial migration, and phagocytosis in a model of acute inflammation.

LPS activation of microglia inhibited the neurite outgrowth of adjacent human

neurons, but the neurite outgrowth reduction could be antagonized by addition of a

CO-donor or induction of HO-1. Since LPS stimulation of microglia affected the

neurite length of adjacent neurons without required cell-cell contact, the effects were

mediated by diffusible factors. One likely candidate is NO which is released in

excessive amounts upon LPS-stimulation of microglia and slowed the neurite

outgrowth of co-cultured neurons.

Finally, in the third part of this thesis evidence has been found that the neurite

outgrowth retarding effect seems to be regulated downstream of NO and CO via the

RhoA (Ras homolog gene family, member A GTPase) / ROCK (Rho-associated

coiled coil forming protein serine/threonine kinase) signaling cascade. Treatment with

RhoA/ROCK inhibiting agents, like the common pain reliever Ibuprofen, greatly

decreased RhoA activation and promoted neurite outgrowth of human neurons.

Conversely, activation of RhoA/ROCK resulted in growth cone collapse.

Taken together, the present thesis provides first insights how cellular responses of

microglia are modulated by the two gases NO and CO and the underlying molecular

signal transduction mechanisms.

Zusammenfassung IX

Zusammenfassung

Hannah Scheiblich

Stickstoffmonoxid (NO)- und Kohlenmonoxid (CO)-vermittelte

Signaltransduktion in einem Ko-Kulturmodell aus Mikroglia und humanen

Modellneuronen

Entzündungsprozesse im Gehirn, die im Wesentlichen mit der Aktivierung spezieller

Immunzellen, den sogenannten Mikroglia einhergehen, spielen bei einer Vielzahl

neurodegenerativer Erkrankungen eine wesentliche Rolle. Mikroglia sind die

residenten Immuneffektorzellen des Zentralnervensystems (ZNS), deren Funktion in

der immunologischen Überwachung des Nervengewebes liegt. Infolge

pathologischer Veränderungen initiieren Mikroglia eine Reihe an zellulären

Immunantworten, die als Verteidigungsstrategie und somit dem Schutz des ZNS

dienen. Dennoch ist die Aktivierung der Mikroglia während inflammatorischer

Prozesse als kritisch zu beurteilen, da eine fehlerhafte Regulierung der mikroglialen

Immunantwort die Pathogenese neurodegenerativer Prozesse verschlimmern kann.

Ziel dieser Arbeit war es, die Relevanz von Stickstoffmonoxid (NO) und

Kohlenmonoxid (CO) sowie ihrer vermittelnden Signaltransduktionswege auf die

Regulierung zellulärer Reaktionen der Mikroglia zu untersuchen.

Um das Migrationsverhalten der Mikroglia sowie die regulatorischen Effekte von NO

und CO auf die Zellmigration zu erforschen, habe ich einen in vitro Versuchsansatz

verwendet, bei dem das Einwandern der Zellen in eine Kratzwunde quantifiziert

wurde. Dabei wurden Zellen der murinen mikroglialen Zelllinie BV-2 sowie primäre

Mikroglia aus der Ratte untersucht und miteinander verglichen. Die Charakterisierung

des Migrationsverhaltens erfolgte durch den Einsatz einer Reihe von

pharmakologischen Wirkstoffen, die den NO/cGMP- sowie den HO-1/CO-Signalweg

auf verschiedenen Ebenen der Signaltransduktion manipulieren. Meine Ergebnisse

weisen deutliche Evidenzen auf, dass NO durch die Aktivierung der löslichen

Guanylatzyklase (sGC) die Bildung von zyklischem Guanosinmonophosphat (cGMP)

stimuliert und so die mikrogliale Zellmigration fördert. Im Gegensatz dazu habe ich

X Zusammenfassung

durch die Aktivierung der Hämoxygenase-1 (HO-1) sowie die Applikation eines CO-

freisetzenden Moleküls (CORM) die HO-1/CO-Kaskade als einen antagonistischen

Modulator der NO/cGMP-Signaltransduktion bei der Regulierung der mikroglialen

Zellmigration identifiziert. Trotz einiger Unterschiede bezüglich der

Konzentrationsbereiche, in denen die pharmakologischen Wirkstoffe eingesetzt

werden mussten, konnte die mikrogliale Zelllinie BV-2 als geeignetes Modellsystem

etabliert werden, um den Einsatz primärer Mikroglia in unseren Versuchsansätzen zu

reduzieren.

In einem weiteren Teil dieser Arbeit habe ich den Fokus auf die regulatorischen

Effekte und die Wechselbeziehung zwischen NO und CO während der Regulierung

der Aktivierung und Phagozytose von Mikroglia gelegt. Meine Ergebnisse zeigen,

dass Stimulation der Mikroglia mittels Lipopolysacchariden (LPS) zu einer deutlich

gesteigerten NO-Freisetzung durch Aktivierung der induzierbaren NO-Synthase

(iNOS) führt. Durch Aktivierung des CO-generierenden Enzyms HO-1 sowie den

Einsatz von CORM konnte die LPS-induzierte iNOS Expression und NO-Produktion

vollständig geblockt werden. Um die Regulierung der Phagozytose der Mikroglia zu

charakterisieren habe ich einen Phagozytose-Assay entwickelt, bei dem Mikroglia

und humane Modellneurone ko-kultiviert wurden. Ich konnte zeigen, dass exogenes

NO nicht nur die iNOS zur endogenen NO-Produktion stimuliert, sondern auch die

Phagozytose-Aktivität der Mikroglia durch Selbststimulation erhöht. Durch Induktion

der HO-1 sowie eine Behandlung mit CORM konnte ich erstmals zeigen, dass der

HO-1/CO-Signalweg die Phagozytose-Aktivität der Mikroglia unter akuten

Entzündungsbedingungen deutlich herunter reguliert.

In einem Versuchsansatz zur Bestimmung des Neuritenwachstums, führte die

Aktivierung der Mikroglia mittels LPS zu einer Inhibition der auswachsenden Neuriten

benachbarter humaner Modellneurone. Diesem inhibitorischen Effekt konnte durch

die Induktion der HO-1 sowie der Zugabe von CORM vollständig entgegengewirkt

werden. Da kein direkter Zell-Zell-Kontakt zwischen Mikroglia und Neuronen für die

Inhibition des Neuritenwachstums erforderlich war, stellten wir die Hypothese auf,

dass diffusionsfähige Faktoren zu diesem negativen Effekt führen müssen. In

weiteren Analysen konnte ich zeigen, dass NO, welches von Mikroglia freigesetzt

Zusammenfassung XI

wird, für den hemmenden Effekt auf das Neuritenwachstum ko-kultivierter Neurone

verantwortlich war.

Im abschließenden Teil dieser These wiesen unsere Ergebnisse deutliche Evidenzen

auf, dass der hemmende Effekt auf das Neuritenwachstum auf einer

nachgeschalteten Ebene der NO- und CO-Signaltransduktion reguliert werden

könnte, nämlich über die RhoA (Ras homolog gene family, member A GTPase) /

ROCK (Rho-associated coiled coil forming protein serine/threonine kinase) -

Signalkaskade. Behandlungen mit Inhibitoren des RhoA/ROCK-Signalwegs, wie

beispielsweise dem gängigen Schmerzmittel Ibuprofen, führten zu einer

verminderten RhoA-Aktivität und förderten so das Neuritenwachstum der humanen

Neurone. Umgekehrt führte die Aktivierung des RhoA/ROCK-Signalwegs zu einem

Zusammenfall des Wachstumskegels.

Die vorliegende Arbeit bietet fundamentale Einblicke in die

Signaltransduktionsmechanismen, die der Regulierung verschiedener zellulärer

Immunantworten von aktivierten Mikroglia via NO und CO zu Grunde liegen. Mit Hilfe

dieses Wissens könnte es möglich werden, die Mikroglia als Schlüssel für die

Entwicklung effizienter Therapiestrategien bei der Behandlung neurodegenerativer

und neuroinflammatorischer Erkrankungen des ZNS zu bestimmen.

Introduction 1

Introduction

Inflammation within the central nervous system (CNS) has been implicated as a

common denominator driving the progression of multiple neurodegenerative

diseases, including Alzheimer’s disease, Parkinson’s disease, multiple sclerosis,

amyotrophic lateral sclerosis, HIV-associated dementia, frontotemporal dementia,

and stroke (for review see: Block and Hong, 2005). There are several cell types that

have been linked to the modulation of inflammation-mediated neurodegeneration, but

to date it is widely accepted that the unregulated response of microglia is associated

with the inflammation-mediated neurodegeneration. As resident immune effector

cells of the CNS, microglia are responsible for brain homeostasis and surveillance

under physiological conditions. However, microglia become rapidly activated in

response to pathological threats (Kreutzberg, 1996).

Under inflammatory conditions, microglia initiate a range of host defense

mechanisms, including the up-regulation of the inducible isoform of the nitric oxide

synthase (iNOS), leading to the generation of high levels of nitric oxide (NO)

(Minghetti and Levi, 1998; Nathan, 1992; Vicente et al., 2001; Vincent, 1994). Even

though microglia-derived NO serves as a cellular defense mechanism at the onset of

inflammation, detrimental side effects may occur as a consequence to the excessive

overproduction of NO by uncontrolled microglial activation. In contrast to NO, the gas

carbon monoxide (CO) has emerged to exert cytoprotective properties (Baranano

and Snyder, 2001; Motterlini and Otterbein, 2010; Soares and Bach, 2009). Although

CO is reported to counteract inflammation and the progression of neurodegeneration,

the knowledge about how CO can regulate cellular characteristics of activated

microglia during these processes is quite limited. Thus, it is of great importance to

understand the signaling pathways that are involved in inflammation and in the

regulation of cellular characteristics driving excessive neuronal loss. Insights into

microglial biology might contribute to pinpoint microglia as a key pharmacological

target for controlling acute inflammatory conditions and neurodegenerative processes

(Block and Hong, 2005; Block et al., 2007; Greter and Merad, 2013; Rock and

Peterson, 2006).

2 Introduction

This thesis examines the regulation of microglial activation including cellular

characteristics such as cell migration, phagocytosis of neurons, and their impact on

neurite outgrowth of adjacent neurons with respect to two major different signaling

cascades. The first section (publication 1, 2) addresses the NO/cGMP signaling

pathway. It is focused on the regulation of microglial cell migration, phagocytic

activity and the impact of microglia on adjacent neurons by using small bioactive

enzyme activators and inhibitors of the NO/cGMP signaling cascade. My doctoral

thesis showed that all of these cellular characteristics of reactive microglia were

regulated by NO. The second section (publication 3) focused on the interrelationship

between NO/cGMP and HO-1/CO signaling in the regulation of microglial features

under inflammatory conditions. Here, our research group showed for the first time

that chemical manipulation of HO-1/CO signaling down-regulates inflammation-

mediated microglial phagocytosis. Moreover, we outlined important initial steps for

designing novel CO-based strategies to medicate excessive inflammation and

microglia-mediated neurodegeneration in the nervous system. In support of the first

two sections, the third section of this thesis (publication 4) presents evidence that

neurite outgrowth of human model neurons is regulated downstream the NO pathway

via the RhoA/ROCK cascade. Here, we showed for the first time that treatment of

human neurons with the commercial pain reliever Ibuprofen enhanced the neurite

growth capacity indicating that inhibition of RhoA/ROCK can overcome the

regeneration inhibitory effects of the CNS environment.

Gatekeepers of the CNS: Microglia in health and disease

Within the CNS, microglia are generally considered as specialized immune

surveillance cells that play a crucial role in mediating brain homeostasis and the

innate immune response against a wide range of pathogenic factors or neuronal

trauma. Depending on their physiological activation level, microglia can be found in

three different characteristic states: (1) resting, ramified microglia found in the healthy

CNS, (2) activated non-phagocytic microglia found in areas of inflammation, and

Introduction 3

(3) reactive, phagocytic microglia found in areas of persisting infection and traumatic

injury (Fig. 1) (Streit et al., 1999).

Under normal physiological conditions microglia exist in a ‘resting' or ramified state

dispersed in all regions of the CNS with cell densities varying between 5% in the

cortex and corpus callosum and 12% in the substantia nigra (Lawson et al., 1990).

Resting microglia are morphologically characterized by a small cell soma with many

thin and highly branched cellular processes extending in all directions. While the

position of the cell soma of resting microglia remains to stay stationary, the

processes constantly move through their microenvironment to scan the brain for

damage or danger signals (Nimmerjahn et al., 2005; Ohsawa and Kohsaka, 2011).

Thereby, every microglia cell covers a separated surveillance territory of about 15-30

µm per cell with a motion speed of up to 1.5 µm/min (Kettenmann and Verkhratsky,

2011). Moreover, microglial processes send out small protrusions which elongate

and retract by 2-3 µm/min (Kettenmann and Verkhratsky, 2011). Thus, the processes

of resting microglia are the most rapidly moving structures within the healthy CNS

enabling microglia to completely scan the brain every several hours and to promptly

respond to environmental changes. To communicate with neighboring cells,

microglial processes directly contact neuronal structures, blood vessels, and

surrounding astrocytes, but avoid contacting adjacent microglia.

For communication and surveillance microglia are equipped with a wealth of cell

surface molecules and pattern-recognition receptors (PRRs) to be able to detect and

discriminate between a wide range of self and non-self stimuli (for review see:

Kierdorf and Prinz, 2013). This enables microglia to sense and respond to an array of

factors, ranging from structural components of exogenous pathogens (pathogen-

associated molecular pattern, PAMP) to abnormal concentrations of soluble and

insoluble endogenous molecules released by damaged cells (damage-associated

molecular pattern, DAMP) (Hanisch and Kettenmann, 2007). The most investigated

and well-studied PRRs are the Toll-like receptor (TLR) family which is known to

consist of 11 members in mammals (Akira and Takeda, 2004). TLRs recognize

structural conserved molecules broadly shared by pathogenic microorganisms like

the lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative

4 Introduction

bacteria. Stimulation initiates a range of host defense mechanisms including dramatic

morphological and functional alterations, by which the cells change from resting

ramified to more reactive cells (Fig. 1) (Hanisch and Kettenmann, 2007; Kreutzberg,

1996; Streit et al., 1999).

Figure 1: Microglial activation states and cellular alterations throughout an activation process.

The “resting” microglia constantly scan their microenvironment for endogenous and exogenous signals

for homeostatic surveillance. Recognition of damage (neuronal) or danger (viral/bacterial) signals such

as nitric oxide (NO) initiates the activation of microglia including dramatic morphological and functional

alterations. Activated or “reactive” microglia become a more amoeboid phenotype to penetrate the

tissue. Depending on the challenging stimuli, reactive microglia are either able to release

neuroprotective factors including carbon monoxide (CO) aiding in brain repair or neurotoxic factors like

Introduction 5

◄ NO that enhance oxidative stress. Persistent activation of reactive microglia further shifts the cells

to “phagocytic microglia” with the ability to engulf damaged neuronal tissue or invading pathogens.

Reactive microglia retract their processes to become a motile amoeboid shape,

penetrate the tissue, gather around the region of the insult and change their reaction

pattern (Kreutzberg, 1996). Depending on their activation stimulus microglia secrete

a myriad of factors that may have either pro- or anti-inflammatory properties (Lee et

al., 2006; Liao, 2004; Liu and Hong, 2003; Morgan et al., 2004; Moss and Bates,

2001; Polazzi et al., 2001). Pro-inflammatory factors mainly exert neurotoxic features

that enhance oxidative stress and induce apoptotic cascades eventually leading to

the progressive loss of neuronal structure and functions. In contrast,

anti-inflammatory responses have rather neuroprotective/neurotrophic activities

aiding in brain repair and regeneration.

However, when a brain insult persists microglia undergo further transformation

changing from reactive to phagocytic microglia (Fig. 1) (Streit et al., 1999) with the

ability to clean up debris, phagocytize invading microorganisms or engulf damaged

neuronal material. In this context, the unregulated activation of microglia and

subsequently the self-sustained cycle of inflammatory responses including the

engulfment of otherwise healthy neuronal structures have been associated with the

pathogenesis of multiple neurodegenerative diseases (Block and Hong, 2005). It is

therefore necessary to understand the conditions and the molecular mechanisms

regulating the balance between the neuroprotective and neurotoxic actions of

microglia.

Cell-to-cell communication signaling

In the present thesis, I will restrict my focus on two gaseous signaling messengers,

nitric oxide and carbon monoxide, which may be crucial regulators in determining the

outcomes of microglial activation and to regulate their reaction pattern under

inflammatory conditions.

6 Introduction

Nitric oxide: the gas with a dual character

Initially, nitric oxide (NO) was discovered as an important gaseous signaling molecule

that plays a key role in neurotransmission, vasodilatation, and development;

however, in the past few years glia-derived NO has been shown to be involved in the

pathogenesis of inflammation followed by excessive tissue damage (Block and Hong,

2005; Block et al., 2007; Liu and Hong, 2003; Neher et al., 2012).

NO is generated by the enzyme nitric oxide synthase (NOS), which converts

L-arginine and O2 in a complex enzymatic reaction to NO and L-citrulline (Fig. 2)

(Nathan, 1992; Vincent, 1994). Under physiological conditions, NO is continuously

produced at rather low levels by the constitutively expressed NOS isoforms. These

isoforms are Ca2+/calmodulin-dependent and are typically expressed in neurons

(neuronal NOS, nNOS or NOS I) and endothelial cells (endothelial NOS, eNOS or

NOS III). Both types of NO synthase (nNOS and eNOS, respectively) produce small

amounts of NO for seconds to minutes which functions as regulator of physiological

activities, such as neurotransmitter and vasodilator (Minghetti and Levi, 1998;

Nathan, 1992; Vincent, 1994). Under pathological conditions, continuously high

levels of NO are produced for hours or even days after induction of the expression of

the inducible isoform of the NOS (inducible NOS, iNOS or NOS II). This Ca2+-

independent NOS is expressed mainly in immune cells including neutrophils,

macrophages, astrocytes, and microglia after stimulation with various types of

pathogenic factors and pro-inflammatory cytokines (Nathan, 1992; Vincent, 1994). In

this context, NO can be considered as a gas with a dual personality (Colasanti and

Suzuki, 2000): on the one hand, NO can exert beneficial effects by acting anti-

bacterial, anti-viral, and tumoricidal (Nathan, 1992); on the other hand, persisting

high levels of NO can cause detrimental side effects resulting in excessive tissue

damage.

The main physiological target enzyme for low concentrations of NO is the soluble

guanylyl cyclase (sGC) (Fig.2) (Garthwaite, 2008). sGC is a heterodimeric protein

comprising a haem-binding region and a catalytic domain (Garthwaite, 2008). Binding

of NO to the haem region leads to the conversion of guanosine triphosphate (GTP) to

cyclic guanosine monophosphate (cGMP) (Garthwaite, 2008; Ignarro, 1990;

Introduction 7

Moncada et al., 1989). cGMP itself activates downstream effectors such as protein

kinases G (PKG) (Hofmann et al., 2006), cyclic nucleotide gated ion channels

(CNGC) (Bender and Beavo, 2006), and cyclic nucleotide phosphodiesterases (PDE)

(Garthwaite, 2008), which in turn modulate the activity of an array of intracellular

signaling molecules, thereby regulating neurotransmission, proliferation, migration,

differentiation, axon outgrowth, and axon guidance.

Figure 2: The NO/cGMP signaling cascade. In immune cells such as microglia the inducible nitric

oxide synthase (iNOS) has been identified to synthesize high levels of NO in response to

immunological stimuli including lipopolysaccharide (LPS). Upon induction iNOS converts L-arginine

and O2 to L-citrulline and NO. NO binds to the soluble guanylyl cyclase (sGC) which leads to the

conversion of guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP). cGMP itself

has the potential to activate downstream effectors such as protein kinase G (PKG), cyclic nucleotide

gated ion channels (CNGC), and cyclic nucleotide phosphodiesterases (PDE), which converts cGMP

to guanosine monophosphate (GMP).

However, several studies focused on cGMP-independent mechanisms of NO’s

actions in the pathogenesis of a number of neurodegenerative diseases

(Madhusoodanan and Murad, 2007). These cGMP-independent actions include

oxidation of thiols, S-nitrosylation, and nitration of proteins which may play a role in

neuronal cell death. Moreover, NO can react with concomitantly produced superoxide

anions to form highly toxic compounds like peroxynitrite (ONOO-) (Brown and Neher,

2010; Neher et al., 2012). The presence of ONOO- then causes DNA damage,

apoptosis and consequently neuronal loss. In addition to this, neurons are

remarkably sensitive to sustained high levels of NO, causing inhibition of neuronal

respiration and subsequently depolarization and glutamate release followed by

excitotoxic death (Bal-Price and Brown, 2001; McNaught and Brown, 1998).

8 Introduction

A better understanding of the molecular mechanisms regulated by NO might provide

an important initial step for designing novel strategies to medicate detrimental side

effects such as neurodegeneration caused by the excessive release of NO.

Carbon monoxide: a novel agent to counteract inflammation?

In the last few years, carbon monoxide (CO) has been emerged as a gaseous

messenger molecule with anti-inflammatory, anti-apoptotic and cytoprotective

properties (Bach, 2005; Baranano and Snyder, 2001; Motterlini and Otterbein, 2010;

Soares and Bach, 2009). However, to date there is only quite limited knowledge

about how CO can affect cellular characteristics of reactive microglia and how CO

exert its anti-inflammatory effects on inflamed cells.

CO is generated by the rate-limiting enzyme heme oxygenase (HO) during the

physiological degradation of heme to biliverdin-IX, ferrous iron (Fe2+), and CO (Fig. 3)

(Choi and Alam, 1996; Maines, 1997; Ryter et al., 2002). Three isoforms of HO have

been characterized: an inducible isoform (HO-1) which is expressed in response to

all kinds of agents and stimuli that have the ability to cause oxidative stress, as well

as inflammogenes such as membrane components of germs (Maines, 1997;

Otterbein and Choi, 2000); a constitutively expressed isoform (HO-2) which is

responsive only to adrenal glucocorticoids (Maines, 1997) and Ca2+/calmodulin

(Boehning et al., 2004); and an elusive and poorly understood catalytically inactive

isoform (HO-3) which is thought to act in oxygen sensing (Hayashi et al., 2004).

However, there is extensive evidence suggesting HO-1 as the major key regulator of

the cytoprotective effects against oxidative stress (Keyse and Tyrrell, 1987, 1989;

Nath et al., 1992) as well as to counteract inflammation (Bach, 2005) and the

progression of neurodegeneration (Syapin, 2008). The mechanisms by which HO-1

exerts its cytoprotective and anti-inflammatory properties are not fully understood, but

implicate a functional role for each of the three catalytic by-products of heme

degradation: biliverdin-IX, Fe2+, and CO (Bach, 2005; Otterbein and Choi, 2000;

Otterbein et al., 2003). To date, there is no other enzyme identified yet that is

affected by such an impressive number of stimuli, as HO-1 does, and that mediates

Introduction 9

the therapeutic actions of other molecules, suggesting that HO-1 functioning as a

“therapeutic amplification funnel” (Fig. 3) (Bach, 2005).

Figure 3: Heme oxygenase-1 (HO-1) as a

therapeutic funnel. HO-1 can be induced

by an impressive number of stimuli amongst

others inflammatory signals, oxidative stress

agents, nitric oxide (NO), carbon monoxide

(CO), and heme. Activation of HO-1 results

in the degradation of heme to biliverdin

(which is consequently reduced to

bilirubin by the biliverdin reductase), CO,

and ferrous iron (Fe2+

). All three components

have the potential to act anti-inflammatory,

or cytoprotective. However, HO-1 has been

implicated to be the real mediator of the

cytoprotective properties. Because HO-1

mediates the actions of other molecules, the

cytoprotective enzyme has been suggested

to function as a therapeutic amplification

funnel.

In addition to its cytoprotective properties, there is increasing evidence for HO-1/CO

signaling as poor activator of the cGMP-synthesizing enzyme sGC. CO stimulates

sGC at ~100-fold lower efficiency than NO, suggesting that the presence of

exogenous CO can modulate the outcomes of NO signaling (Baranano and Snyder,

2001; Kharitonov et al., 1995; Knipp and Bicker, 2009).

A better understanding about how HO-1/CO signaling could develop its

cytoprotective properties on inflamed microglia might provide important insights in

regulating neurodegenerative processes in the nervous system.

Feedback between nitric oxide and carbon monoxide generation

It is becoming increasingly evident that iNOS/NO and HO-1/CO signaling are tightly

linked, mutually affect the induction of each other, and share many of their chemical

10 Introduction

properties (Maines, 1997). The fact, that NO causes HO-1 up-regulation while in

parallel HO-1 induction resulted in the down-regulation of iNOS, led various scientists

to investigate the interrelationship of NO and CO under inflammatory conditions

(Fig. 4). Inflammation is usually accompanied by the induction of iNOS via the

nuclear factor NF-ƙB (nuclear factor kappa-light-chain-enhancer of activated B cells)

and subsequently the generation of NO (Lee et al., 2004; Wang et al., 2002). NO

itself causes oxidative stress, which has been implicated in the activation of the

transcription factor Nrf2 (nuclear factor-erythroid 2-related factor 2) (Terazawa et al.,

2013). Nrf2 regulates the expression of various cytoprotective genes including HO-1,

leading to an enhanced HO-1 induction (Itoh et al., 1997). In addition to Nrf2, NF-ƙB

is an important component of HO-1 induction in response to a diversity of pro-

inflammatory stimuli (Lee and Suk, 2007; Lu et al., 2010).

Figure 4: Feedback between nitric oxide (NO) and carbon monoxide (CO) generation.

Lipopolysaccharide (LPS) induces inducible NO synthase (iNOS) expression and NO production via

NF-ƙB (nuclear factor kappa-light-chain-enhancer of activated B cells). NO itself causes the activation

of Nrf2 (nuclear factor-erythroid 2-related factor 2) which regulates the expression of the heme

oxygenase-1 (HO-1). In parallel, LPS activates HO-1 via NF-ƙB. Induction of HO-1 leads to the

degradation of heme to ferrous iron (Fe2+

), CO and Biliverdin (which is converted to Bilirubin) with all

three by-products having the capacity to block iNOS expression and subsequently to reduce NO

production.

Introduction 11

Activation of HO-1 leads to the degradation of heme to biliverdin-IX, Fe2+, and CO

with all three components having the capacity to block iNOS expression and with this

to prevent excessive NO production (Liu et al., 2003; Min et al., 2006; Srisook et al.,

2006; Wang et al., 2004) In addition, Fe2+ is thought to enhance the cellular iron

export (Ferris et al., 1999). Biliverdin, which is rapidly converted to bilirubin by

biliverdin reductase has been reported to act as an potent antioxidant (Stocker et al.,

1987). And CO, the main by-product of heme-degradation, has been shown to exert

its anti-inflammatory effects by decreasing the production of inflammatory mediators

in macrophages and microglia exposed to bacterial endotoxins (Bani-Hani et al.,

2006a, 2006b; Sawle et al., 2005). Intriguingly, pharmacological blockage of HO-1

amplifies the inflammatory response of LPS-treated macrophages whereas the

application of a CO-donor reversed this effect (Sawle et al., 2005).

12 Aims of the dissertation

Aims of the dissertation

Inflammation within the CNS is usually characterized by the activation of microglia

and has been closely associated with the pathogenesis of several neurological

disorders with a neurodegenerative component. Microglia play a dual role in

mediating the immune response after infections or injuries. Depending on their

activation state, they are either able to amplify inflammatory conditions by the release

of pro-inflammatory molecules, or to protect the neuronal tissue by the release of

anti-inflammatory cytokines and the engulfment of infected cells. Insights into

mechanisms of activation, migration, phagocytosis, and neuron-interaction may help

to pinpoint microglia as a key pharmacological target in the treatment of

neurodegeneration.

Specific aims of this thesis were:

(1) to introduce the murine microglial cell line BV-2 as a useful model system for

primary microglia

(2) to develop an in vitro cell migration assay for the investigation whether

NO/cGMP signaling could modulate microglial movement and to test for

potential interactions with the HO-1/CO signaling cascade

(3) to develop an in vitro phagocytosis assay with microglia and human model

neurons to quantify possible anti-inflammatory and cytoprotective properties of

the HO-1/CO cascade and to determine the effects of NO signaling

(4) to devise a suitable co-culture assay to investigate potential crosstalk between

microglial activation, pharmacological manipulation of activated microglia, and

its impact on neurite outgrowth of developing neurons

(5) and to elucidate the mechanisms contributing to these effects

Publications 13

Publications

This thesis was prepared as a cumulative dissertation comprising four original

publications. Three of these publications are first-authored. For a better

understanding, articles are not presented in their chronological order of publication

date.

Authors’ contributions

1) Scheiblich H, Roloff F, Singh V, Stangel M, Stern M, Bicker G (2014) Nitric

oxide / cyclic GMP regulates motility of a microglial cell line and primary

microglia, Brain Research 1564: 9-21. DOI: 10.1016/j.brainres.2014.03.048

designed experiments: HS, MS, GB; performed experiments: HS; isolated

primary microglia: VS; analyzed data: HS, GB; wrote the article: HS (with input

from GB); corrected and improved the manuscript: all authors

Some data contributing to this publication have already been collected during my

masters’ thesis (2012) entitled “Zellmigrations-Assay an primären Mikroglia und der

mikroglialen Zelllinie BV-2” at the University of Veterinary Medicine Hannover,

Germany.

2) Scheiblich H, Bicker G (2015) Nitric oxide regulates antagonistically

phagocytic and neurite outgrowth inhibiting capacities of microglia,

Developmental Neurobiology, accepted. DOI: 10.1002/dneu.22333

designed experiments: HS, GB; performed experiments: HS; analyzed data:

HS, GB; wrote the article: HS (with input from GB); corrected and improved

the manuscript: all authors

14 Publications

3) Scheiblich H, Bicker G (2015) Regulation of microglial migration,

phagocytosis, and neurite outgrowth by HO-1/CO signaling,

Developmental Neurobiology 75(8): 854-876. DOI: 10.1002/dneu.22253

designed experiments: HS, GB; performed experiments: HS; analyzed data:

HS, GB; wrote the article: HS (with input from GB); corrected and improved

the manuscript: all authors

4) Roloff F, Scheiblich H, Dewitz C, Dempewolf S, Stern M, Bicker G (2015)

Enhanced neurite outgrowth of human model (NT2) neurons by small-

molecule inhibitors of Rho/ROCK signaling, PLoS ONE 10(2): e0118536.

DOI: 10.1371/journal.pone.0118536

designed experiments: FR, GB; performed experiments: FR, HS (RhoA pull-

down activation assay and western blot analysis), CD, SD; analyzed data: FR,

HS (RhoA pull-down activation assay and western blot analysis), CD, SD, GB;

wrote the article: FR (with input from GB); corrected and improved the

manuscript: all authors

Publications 15

Publication 1

Nitric oxide / cyclic GMP regulates motility of a microglial cell line

and primary microglia

Hannah Scheiblich, Frank Roloff, Vikramjeet Singh, Martin Stangel, Michael Stern,

Gerd Bicker (2014) Nitric oxide / cyclic GMP regulates motility of a microglial

cell line and primary microglia. Brain Research 1564: 9-21.

http://www.sciencedirect.com/science/article/pii/S0006899314004521

DOI: 10.1016/j.brainres.2014.03.048

Abstract

Microglia are the resident immune cells of the brain, which become rapidly activated and

migrate to the site of insult in brain infection and disease. Activated microglia generate large

amounts of the highly reactive messenger molecule nitric oxide (NO). NO is able to raise

cyclic GMP levels via binding to soluble guanylyl cyclase. We investigated potential

mechanistic links between inflammation, NO signaling, and microglial migration. To monitor

cell migration, we used a scratch wound assay and compared results obtained in the BV-2

microglial line to primary microglia. Incubation with lipopolysaccharide (LPS) as stimulator of

acute inflammatory processes enhanced migration of both microglial cell types. LPS

activated NO production in BV-2 cells and application of an NO donor increased BV-2 cell

migration while an NO scavenger reduced motility. Pharmacological inhibition of soluble

guanylyl cyclase and the resulting decrease in motility can be rescued by a membrane

permeant analog of cGMP. Despite differences in the threshold towards stimulation with the

chemical agents, both BV-2 cells and primary microglia react in a similar way. The important

role of NO/cGMP as positive regulator of microglial migration, the downstream targets of the

signaling cascade, and resulting cytoskeletal changes can be conveniently investigated in a

microglial cell line.

16 Publications

Publication 2

Nitric oxide regulates antagonistically phagocytic and neurite

outgrowth inhibiting capacities of microglia

Hannah Scheiblich, Gerd Bicker (2015) Nitric oxide regulates antagonistically

phagocytic and neurite outgrowth inhibiting capacities of microglia.

Developmental Neurobiology, accepted for publication.

http://onlinelibrary.wiley.com/doi/10.1002/dneu.22333/abstract

DOI: 10.1002/dneu.22333

Abstract

Traumatic injury or the pathogenesis of some neurological disorders is accompanied by

inflammatory cellular mechanisms, mainly resulting from the activation of CNS resident

microglia. Under inflammatory conditions, microglia upregulate the inducible isoform of NOS

(iNOS), leading to the production of high concentrations of the radical molecule nitric oxide

(NO). At the onset of inflammation, high levels of microglial-derived NO may serve as a

cellular defense mechanism helping to clear the damaged tissue and combat infection of the

CNS by invading pathogens. However, the excessive overproduction of NO by activated

microglia has been suggested to govern the inflammation-mediated neuronal loss causing

eventually complete neurodegeneration.

Here, we investigated how NO influences phagocytosis of neuronal debris by BV-2 microglia,

and how neurite outgrowth of human NT2 model neurons is affected by microglial-derived

NO. The presence of NO greatly increased microglial phagocytic capacity in a model of acute

inflammation comprising lipopolysaccharide (LPS)-activated microglia and apoptotic neurons.

Chemical manipulations suggested that NO upregulates phagocytosis independently of the

sGC/cGMP pathway. Using a transwell system, we showed that reactive microglia inhibit

neurite outgrowth of human neurons via the generation of large amounts of NO over effective

distances in the millimeter range. Application of a NOS blocker prevented the LPS-induced

NO production, totally reversed the inhibitory effect of microglia on neurite outgrowth, but

reduced the engulfment of neuronal debris. Our results indicate that a rather simple notion of

treating excessive inflammation in the CNS by NO synthesis blocking agents has to consider

functionally antagonistic microglial cell responses during pharmaceutic therapy.

Publications 17

Publication 3

Regulation of microglial migration, phagocytosis and neurite

outgrowth by HO-1/CO signaling

Hannah Scheiblich, Gerd Bicker (2015) Regulation of microglial migration,

phagocytosis and neurite outgrowth by HO-1/CO signaling. Developmental

Neurobiology 75(8): 854-876.

http://onlinelibrary.wiley.com/doi/10.1002/dneu.22253/abstract

DOI: 10.1002/dneu.22253

Abstract

Clearance of infected and apoptotic neuronal corpses during inflammatory conditions is a

fundamental process to create a favorable environment for neuronal recovery. Microglia are

the resident immune cells and the predominant phagocytic cells of the CNS, showing a

multitude of cellular responses upon activation. Here, we investigated in functional assays

how the CO generating enzyme heme oxygenase 1 (HO-1) influences BV-2 microglial

migration, clearance of debris, and neurite outgrowth of human NT2 neurons. Stimulation of

HO-1 activity attenuated microglial migration in a scratch wound assay, and phagocytosis in

a cell culture model of acute inflammation comprising lipopolysaccharide (LPS)-activated

microglia and apoptosis-induced neurons. Application of a CO donor prevented the

production of NO during LPS stimulation, and reduced microglial migration and engulfment of

neuronal debris. LPS-activated microglia inhibited neurite elongation of human neurons

without requiring direct cell-cell surface contact. The inhibition of neurite outgrowth was

totally reversed by application of exogenous CO or increased internal CO production through

supply of the substrate hemin to HO. Our results point towards a vital cytoprotective role of

HO-1/CO signaling after microglial activation. In addition, they support a therapeutic potential

of CO releasing chemical agents in the treatment of excessive inflammatory conditions in the

CNS.

18 Publications

Publication 4

Enhanced neurite outgrowth of human model (NT2) neurons by

small-molecule inhibitors of Rho/ROCK signaling

Frank Roloff, Hannah Scheiblich, Carola Dewitz, Silke Dempewolf, Michael Stern,

Gerd Bicker (2015) Enhanced neurite outgrowth of human model (NT2) neurons

by small-molecule inhibitors of Rho/ROCK signaling. PLoS ONE 10(2):

e0118536.

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0118536

DOI: 10.1371/journal.pone.0118536

Abstract

Axonal injury in the adult human central nervous system often results in loss of sensation

and motor functions. Promoting regeneration of severed axons requires the inactivation of

growth inhibitory influences from the tissue environment and stimulation of the neuron

intrinsic growth potential. Especially glial cell derived factors, such as chondroitin sulfate

proteoglycans, Nogo-A, myelin-associated glycoprotein, and myelin in general, prevent axon

regeneration. Most of the glial growth inhibiting factors converge onto the Rho/ROCK

signaling pathway in neurons. Although conditions in the injured nervous system are clearly

different from those during neurite outgrowth in vitro, here we use a chemical approach to

manipulate Rho/ROCK signalling with small-molecule agents to encourage neurite outgrowth

in cell culture. The development of therapeutic treatments requires drug testing not only on

neurons of experimental animals, but also on human neurons. Using human NT2 model

neurons, we demonstrate that the pain reliever Ibuprofen decreases RhoA (Ras homolog

gene family, member A GTPase) activation and promotes neurite growth. Inhibition of the

downstream effector Rho kinase by the drug Y-27632 results in a strong increase in neurite

outgrowth. Conversely, activation of the Rho pathway by lysophosphatidic acid results in

growth cone collapse and eventually to neurite retraction. Finally, we show that blocking of

Rho kinase, but not RhoA results in an increase in neurons bearing neurites. Due to its anti-

inflammatory and neurite growth promoting action, the use of a pharmacological treatment of

damaged neural tissue with Ibuprofen should be explored.

Discussion 19

Discussion

This thesis provides a comprehensive overview how the two gaseous messengers,

NO and CO, influence certain aspects of microglia biology. For reasons of

importance to medical treatment of neurodegenerative processes, I took advantage

of performing experimental manipulations of the NO and CO signaling cascades in

cell culture assays. I focused on the possibility to control different characteristics of

microglia under basal conditions and after LPS-induced activation. Moreover,

molecular mechanisms contributing to the detrimental effects of activated microglia

could be elucidated.

Key findings of this work are:

the BV-2 microglial cell line is a useful model for primary microglia in assays

where robust microglial cell behavior is required

NO/cGMP signaling positively regulates migration of primary microglia and

BV-2 cells and causes cytoskeletal changes in both cell types

migratory response of microglia is dually regulated by antagonistic interacting

iNOS/NO/cGMP and HO-1/CO/cGMP signal transduction pathways

NO signaling enhances phagocytic activity of microglia in a co-culture system

with apoptosis-induced human NT2 (Ntera2/D1 precursor cells) model

neurons

stimulation of HO-1/CO signaling prevents the LPS-induced NO production

and may thus be a powerful modulator of inflammation-activated microglia

chemical manipulation of HO-1/CO signaling down-regulates inflammation-

mediated microglial phagocytosis and represent an important initial step for

designing novel CO-donor based strategies to medicate excessive

inflammation in the nervous system

20 Discussion

LPS-activated microglia inhibit neurite elongation of human neurons without

required cell-cell surface contact via the release of NO

stimulation of HO-1/CO signaling and inhibition of iNOS induction totally

reverse the detrimental effects of LPS-activated microglia on neurite outgrowth

of the model neurons and may thus be helpful to facilitate neurite outgrowth

for the repair of injured neural connections

in vitro treatment of developing model neurons with RhoA/ROCK inhibiting

agents greatly increase neurite outgrowth, indicating that the regulation is

caused downstream of the NO/cGMP pathway

The following discussion will focus on these outcomes and provide some

perspectives for future experiments.

Discussion 21

BV-2 cells as model system for primary microglia

The isolation of primary microglia from rat brains is a time consuming procedure

yielding rather low cell numbers with a limited proliferation capacity for in vitro

culturing. The use of a microglial cell line might thus serve as a potential method to

replace in vivo experiments and to reduce the high impact of animal consumption in

research (Henn et al., 2009). Most work on microglial activation, cell signaling, and

function has thus been performed using rapidly proliferating microglial cell lines such

as N9 (Corradin et al., 1993) and BV-2 (Blasi et al., 1990).

In this study, I used the in vitro scratch wound assay to introduce the immortalized

murine microglial cell line BV-2 (Blasi et al., 1990) as a valid substitute for primary

microglia (Scheiblich et al., 2014). Cell migration behavior of BV-2 cells versus

primary microglia was analyzed by chemical manipulation of the NO/cGMP signaling

cascade (Fig. 2). I found, that despite differences in the threshold towards stimulation

with the chemical agents, both BV-2 cells and primary microglia reacted in a similar

way upon manipulation (Scheiblich et al., 2014). Moreover, the reaction patterns of

our two microglial cell types were in line with several other cell culture and in vivo

studies investigating the effects of NO/cGMP on microglial cell motility upon chemical

manipulation (Chen et al., 2000; Dibaj et al., 2010; Duan et al., 2009; Haynes et al.,

2006; Ohsawa et al., 2007). In addition to their migration behavior, I found that BV-2

cells release NO time- and dose-dependently upon LPS challenge similar than

primary microglia (Blasi et al., 1990; Henn et al., 2009; Horvath et al., 2008; Stansley

et al., 2012). In another part of this approach, I showed that BV-2 cells have the

potential to engulf apoptosis-induced neurons and to influence neurite outgrowth of

human NT2 model neurons (Scheiblich and Bicker, 2015a, 2015b). Nevertheless, for

a straightforward comparison of both cell types, it should be kept in mind that primary

microglia are a heterogeneous population isolated from different brain regions with

diverse reaction patterns, whereas BV-2 cells perform more uniformly (see

discussion in Scheiblich et al., 2014).

However, the robust response in our in vitro assays, the cellular homogeneity, and

the unlimited proliferation potential make the BV-2 microglial cell line an ideal model

for the investigation of microglial functions during acute inflammatory conditions.

22 Discussion

Microglial activation: why could carbon monoxide be beneficial for

the inflamed brain?

One of the first responses after a pathological event is the activation of microglia

which are prepared to recognize and discriminate between a wide range of

pathogenic stimuli with a high level of fine-tuned responsiveness (Hanisch and

Kettenmann, 2007). In general, microglial activation is thought to be protective to the

brain parenchyma. However, many studies have concluded that detrimental side

effects may eventuate in collateral damage following excessive periods of microglial

activation (Bal-Price and Brown, 2001; Banati et al., 1993; Bolaños et al., 1997). The

mechanisms by which reactive microglia drive neurodegenerative processes are only

partly known, but implicate the release of microglia-derived NO (Bal-Price and

Brown, 2001; Bolaños et al., 1997; Chao et al., 1996; Loihl and Murphy, 1998).

In line with previous studies (Arimoto and Bing, 2003; Boje and Arora, 1992; Brown,

2007; Hunot et al., 1996), results of the present thesis support evidence that

microglial activation, the induction of iNOS and the sustained release of NO might

potentially play a role in regulating neurodegenerative conditions. I found that

treatment of cultured microglia with the inflammogen LPS not only increased the

protein expression of iNOS and subsequently the microglia-derived NO production

(Fig. 5), but also up-regulated cellular characteristics of microglia, such as cell

migration, phagocytic activity, and neurite growth retarding effects on adjacent

neurons (Scheiblich and Bicker, 2015a, 2015b; Scheiblich et al., 2014). By contrast,

inhibition of iNOS expression and NO production provided more neuroprotective

effects by down-regulating these cellular characteristics (Scheiblich and Bicker,

2015b).

During the last decades the CO generating enzyme HO-1 has been emerged as a

novel target to counteract inflammation and the progression of neurodegenerative

diseases (Baranano and Snyder, 2001; Motterlini and Otterbein, 2010; Soares and

Bach, 2009; Syapin, 2008). I found, that in LPS-activated microglia, the chemical

manipulation of the HO-1 signaling cascade inhibited the induction of the iNOS

protein expression and subsequently decreased the NO production (Fig. 5)

(Scheiblich and Bicker, 2015a). In addition, chemical manipulation of HO-1/CO

Discussion 23

signaling down-regulated cellular characteristics of reactive microglia including cell

migration, phagocytic activity, and the retarding effects on neurite outgrowth of co-

cultured neurons (Scheiblich and Bicker, 2015a). Intriguingly, treatment with LPS or

exogenous NO induced the expression of HO-1 (Scheiblich and Bicker, 2015a;

Terazawa et al., 2013), suggesting reciprocal interactions between iNOS and HO-1

mediated signal transduction (Fig. 5). In support of our observations, it has been

already demonstrated that all three catalytic by-product (biliverdin, Fe2+, and CO)

arisen from HO-1 degradation of heme have the potential to block iNOS expression

and subsequently NO production (Liu et al., 2003; Min et al., 2006; Srisook et al.,

2006; Wang et al., 2004). Nevertheless, CO seems to be the main inhibitor of iNOS

expression and NO generation since CO has the potential to bind to the heme group

of iNOS and with that to inhibit the electron transfer reaction which is required for the

production of NO (Turcanu et al., 1998a, 1998b).

Figure 5: Interaction between nitric oxide (NO) and carbon monoxide (CO) signaling in

microglia. Chemical manipulation of HO-1/CO signal transduction suggests reciprocal interactions

with iNOS/NO signaling. Upon detecting inflammogenes such as lipopolysaccharide (LPS) microglial

inducible NO synthase (iNOS) synthesize high levels of NO. Similar to iNOS, heme oxygenase-1

(HO-1) is induced by LPS to produce CO. Moreover, HO-1 can be activated by application of the HO-

substrate hemin. The presence of CO markedly decreases iNOS induction and subsequently NO

production. Representative western blots of microglia treated for 24 h with different chemical

compounds interfering with NO and CO signal transduction revealed our findings.

Findings of the present work support a therapeutic potential of NO synthesis blocking

agents and CO-releasing molecules (CORMs) to medicate excessive inflammation in

24 Discussion

the nervous system. However, for a straightforward statement one must ask whether

LPS-induced microglial activation in culture accurately reflect microglial functions in

the intact nervous system (Hanisch and Kettenmann, 2007). LPS stimulation has

long been considered as the gold standard for microglial activation, but stimulation

with the bacterial endotoxin results in a massive all-or-none defense reaction. In the

CNS the precise nature of microglial activation is usually context dependent and

implicates the adaptation to even tiny environmental changes via many sub-states on

the way from resting to phagocytic microglia. Thus, the development of suitable

therapies requires further investigation in relevant in vitro and in vivo models of

inflammation-mediated neurodegeneration.

Reciprocity of nitric oxide and carbon monoxide in regulating

microglial cell migration

Upon activation microglial migration from their resting location in the CNS to the site

of insult is an essential component of the cellular response under pathological

conditions (Chen et al., 2000; Dibaj et al., 2010; Duan et al., 2009; Kreutzberg,

1996). A series of studies recently addressed various molecules initiating microglial

movement in abnormal concentrations including adenosine triphosphate (ATP) (Duan

et al., 2009), cannabinoids (Walter et al., 2003), chemokines (Cross and Woodroofe,

1999), and lysophosphatidic acid (LPA) (Schilling et al., 2004).

We and others found that NO released by degenerating neurons and activated

microglia itself acts as a modulator and chemoattractant to microglial migration

resulting in the recruitment of the cells toward the insult (Chen et al., 2000; Dibaj et

al., 2010; Duan et al., 2009; Haynes et al., 2006; Ohsawa et al., 2007; Scheiblich et

al., 2014). Interestingly, low concentrations of NO enhanced the movement of

migrating microglia while high concentrations acted as a stop signal to may cause

accumulation at the lesion site. Application of small bioactive enzyme activators and

inhibitors of the NO/cGMP signaling cascade indicated that NO positively modulates

migration of microglia via cGMP (Fig. 6) (Scheiblich et al., 2014). However, the

outcome of cellular signal transduction is complex because multiple signaling

Discussion 25

pathways could operate in parallel and may interact with each other at different levels

of the downstream cascade (Fig. 6). To advance our understanding how CO

signaling can exert its anti-inflammatory and cytoprotective effects during microglial

recruitment, I investigated whether microglial motility could be modulated by chemical

manipulation of the intracellular HO-1/CO signaling cascade. In my experiments,

application of a chemical CO-donor (CORM-II) and stimulation of HO-1 (with the

HO-1 substrate hemin) implicated CO as an antagonist to NO in the modulation of

microglial cell migration (Scheiblich and Bicker, 2015a).

Figure 6: Microglial migration is

antagonistically regulated by nitric

oxide (NO) and carbon monoxide (CO).

Similar to NO, CO binds to the soluble

guanylyl cyclase (sGC) and regulates the

conversion of guanosine triphosphate

(GTP) to cyclic guanosine monophosphate

(cGMP) but with ~100-fold lower

efficiency. cGMP leads to the activation of

the protein kinase G (PKG) which in turn

results in cytoskeletal reorganization

followed by increased cell motility.

Similar to NO, the release of endogenous CO seems to be able to activate the sGC

but with ~100-fold lower efficiency. Thus, the binding of CO to sGC might down-

regulate the efficacy of NO activation by a competitive mechanism (Knipp and Bicker,

2009). In addition to CO, biliverdin another by-product of heme degradation has been

shown to block the NO-induced activity of sGC (Koglin and Behrends, 2002). Thus,

the induction of HO-1 and subsequently the presence of CO and biliverdin can

modulate the outcome of NO/cGMP signal transduction (Baranano and Snyder,

2001; Kharitonov et al., 1995; Knipp and Bicker, 2009; Scheiblich and Bicker, 2015a).

Since our experimental paradigm allow for optical imaging of migrating microglia, we

can now investigate essential links between the therapeutic benefit of drugs to

counteract excessive microglial activation and the resulting migration behavior.

26 Discussion

Antagonistic regulation of microglial phagocytosis by nitric oxide

and carbon monoxide

A key function of microglia upon reaching the inflammatory epicenter, but also in

non-inflammatory environments is debris clearance (Neumann et al., 2009), including

phagocytosis of apoptotic neurons. However, the inflammation-related microglia-

mediated excessive uptake of healthy neurons is considered to be a characteristic

feature in the onset and progression of neurodegenerative processes (Glass et al.,

2010; Lucin and Wyss-Coray, 2009). In general, microglial phagocytosis has been

summarized in a three-step model: find-me, eat-me, and digest-me (for review see:

Savill et al., 2002; Sierra et al., 2013). As mentioned earlier in this thesis, the first

step (‘find-me‘) is triggered by signals released from damaged neurons including NO

to attract microglia (see discussion in Scheiblich et al., 2014). The second step (‘eat-

me’) includes the recognition and engulfment of the apoptotic cell (see discussion in

Scheiblich and Bicker, 2015a, 2015b), triggered by the interaction of microglial

receptors and their ligands on the membrane of the target cell, such as

phosphatidylserine (PS) (Neher et al., 2012). The third step (‘digest-me’) implicates

the full degradation of the apoptotic cell in the phagolysosome (Sierra et al., 2013).

The mechanisms regulating the engulfment of healthy neurons by inflamed microglia

are not completely understood but implicate a significant role for NO (for review see:

Block and Hong, 2005; Brown, 2007). In line with this, results from the present thesis

argue for a regulating impact of NO as enhancer of microglial phagocytosis (Fig. 7)

(Scheiblich and Bicker, 2015b). I found that beside LPS-stimulated microglia,

microglia challenged with exogenous NO increased their NO production via iNOS

induction. The presence of NO enhanced the phagocytic activity through

self-stimulation independent of the source of NO (Scheiblich and Bicker, 2015b).

Strong evidence has accumulated that the activation of microglia through Toll-like

receptor 4 results in the induction of iNOS and the assembly of the phagocytic

oxidase (PHOX), resulting in the production of NO and superoxide (O2-), respectively

(Brown and Neher, 2010; Neher et al., 2012). NO and O2- subsequently react to form

ONOO- which causes apoptosis and consequently a disrupted lipid asymmetry of the

plasma membrane followed by the externalization of PS to the outer membrane

Discussion 27

leaflet of neurons (Neher et al., 2012). The process which is known as scrambling

has been considered a characteristic feature of apoptosis and requires the activity of

a membrane protein called scramblase (for review see: Mariño and Kroemer, 2013).

Xk-related protein 8 (Xkr8) has recently been identified as the enzyme responsible

for the apoptosis-induced lipid scrambling (Suzuki et al., 2013). The mechanisms of

apoptosis-induced PS exposure is poorly understood, but implicated the presence of

caspase recognition sites of Xkr8 and caspase cleavage. Since ONOO--induced

apoptosis has been reported to activate several caspases (Zhuang and Simon,

2000), it is likely that similar mechanisms regulate microglia-mediated PS exposure

of adjacent and otherwise healthy neurons. The exposure of PS to the outer

membrane leaflet of neurons is then recognized by microglia as an eat-me signal,

thereby causing engulfment (Neher et al., 2012). Inhibition of iNOS and PHOX

prevented the formation of ONOO-, exposure of PS and phagocytosis (Kumar et al.,

2014; Mander and Brown, 2005; Scheiblich and Bicker, 2015b).

Figure 7: Nitric oxide (NO)-mediated microglial (green) phagocytosis of neuronal (magenta)

debris is down-regulated by the induction of heme oxygenase-1 (HO-1) / carbon monoxide (CO)

signaling. Detection of inflammogenes such as lipopolysaccharide (LPS) caused the increased

expression of the inducible NO synthase (iNOS) and activation of the phagocytic oxidase to produce

NO and superoxide (O2-), respectively. NO and O2

- subsequently react to form peroxynitrite (ONOO

-)

leading to the exposure of phosphatidylserine (PS) in neurons. Recognition of PS on the outer

membrane leaflet of neurons and binding to the microglial phosphatidylserine-receptor (PS-R) results

in the engulfment of the neuron. Induction of HO-1/CO signaling inhibits LPS-induced iNOS induction

and NO production resulting in a blocked phagocytic activity.

28 Discussion

Moreover, to the best of current knowledge, it was shown for the first time that the

phagocytosis enhancing effect of microglia activation could be overcome by the

induction of HO-1/CO signaling and treatment with a CO-releasing molecule (CORM)

(Scheiblich and Bicker, 2015a). Since HO-1/CO signaling effectively prevented LPS-

induced iNOS expression and subsequently NO production of microglia, my results

suggest a functional antagonism between NO and CO signaling in the regulation of

microglial phagocytosis (Fig. 7).

However, beside its detrimental side effects NO might aid in the removal of already

damaged cells and in initiating repair mechanisms under injured conditions. There is

therefore the need to carefully evaluate the exact time frame in which NO acts

beneficial by enhancing the clearance of wounded nervous tissue to promote

functional recovery, and in which NO exerts more detrimental side effects such as

excessive inflammation and loss of healthy neurons.

Neurite outgrowth of human neurons is regulated by microglia

Traumatic damage to the nervous system including spinal cord injury (SCI) causes

extensive inflammation and the invasion of microglia in and around the affected

regions. Previous work has shown, that activation and accumulation of microglia

around the epicenter of the lesion is thought to be involved in the quite limited axonal

recovery of the CNS (Kitayama et al., 2011; Popovich et al., 1999). The molecular

mechanisms blocking neurite regrowth and regeneration are not completely

understood but they implicate microglia-derived substances acting as axon growth

inhibitors.

Results of the present thesis support evidence that activation of microglia attenuate

neurite outgrowth of co-cultured neurons without direct cell-cell surface contact.

Thus, we hypothesized that microglia-derived secreted molecules must be

responsible for the poor axonal elongation (Fig. 8 A). By performing experimental

manipulations I found that the induction of microglial iNOS and the presence of NO

were responsible for blocking neurite growth following growth cone collapse

(Scheiblich and Bicker, 2015b). Intriguingly, inhibition of iNOS/NO production via

Discussion 29

induction of HO-1/CO signaling not only attenuated the inflammatory response of

reactive microglia (see discussion before) but also allowed for neurite outgrowth

(Scheiblich and Bicker, 2015a). In support of these observations we found, that the

regulation of neurite outgrowth is caused downstream the NO pathway (Fig. 8 B)

(Roloff et al., 2015).

Figure 8: Neurite outgrowth of human model neurons (magenta) is regulated by microglia

(green). (A) Microglial activation result in the induction of the inducible nitric oxide synthase (iNOS)

and subsequently in the release of nitric oxide (NO). The presence of NO blocks neurite outgrowth of

the neurons. However, induction of heme oxygenase-1 (HO-1) and the release of carbon monoxide

(CO) rescue the detrimental effects of microglia-derived NO on neurite outgrowth. (B) Sine both, NO

and CO, have the potential to bind to the soluble guanylyl cyclase (sGC) and thus to regulate the

conversion of guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP) by the

protein kinase G (PKG) it is likely that considerable parts of neurite growth are regulated downstream

the sGC/cGMP cascade. In support of this, treatment with the commercial pain reliever Ibuprofen

(RhoA (Ras homolog gene family, member A GTPase) and Rho kinase ROCK (Rho-associated coiled

coil forming protein serine/threonine kinase) inhibiting agent) greatly increased neurite length of the

human model neurons while activation of RhoA/ROCK result in growth cone collapse followed by the

inhibition of neurite growth.

We and others revealed a pivotal role for RhoA (Ras homolog gene family, member

A GTPase) and the Rho kinase ROCK (Rho-associated coiled coil forming protein

serine/threonine kinase) signaling on neurite outgrowth of neurons (Boomkamp et al.,

2012; DeGeer and Lamarche-Vane, 2013; Tönges et al., 2011; Wu et al., 2009).

Treatment with RhoA/ROCK inhibiting agents increased neurite length of the human

30 Discussion

model neurons (Roloff et al., 2015). Moreover, activation of RhoA caused

cytoskeletal changes eventually leading to a growth cone collapse and suppression

of axonal re-extension (Gallo and Letourneau, 2004). Since PKG, which is activated

downstream the NO/cGMP cascade, regulates the expression and phosphorylation

of RhoA (Sauzeau et al., 2003), it is possible that reactive microglia mediate their

harmful effects via NO/cGMP/PKG - RhoA/ROCK activation (Fig. 8).

There are, however, also indications that NO-initiated growth cone collapse, axon

retraction and reconfiguration of axonal microtubules are not mediated via

cGMP/PKG (Ernst et al., 2000; He et al., 2002; Hess et al., 1993). A key event in NO

signaling to the axonal cytoskeleton is triggered through the S-nitrosylation of the

microtubule-associated protein 1B (MAP1B) (Stroissnigg et al., 2007) which is highly

expressed during development and regeneration (Gordon-Weeks and Fischer, 2000;

Soares et al., 2002). S-nitrosylation of MAP1B increases its microtubule binding and

inhibits dynein actions leading to a reduction of neurite extension force (Stroissnigg

et al., 2007)

Even though microglia-mediated neurite outgrowth inhibition is thought to be partially

responsible for the limited axonal recovery, it may initially be advantageous to

prevent the ingrowth of neuronal processes into the hostile environment of wounded

nervous tissue. Manipulation of microglial activation might thus offer a potential

pharmacological target for controlling neuronal repair under acute

inflammatory/traumatic conditions, but the exact time frame for a potential treatment

has to be carefully evaluated (Block and Hong, 2005; Block et al., 2007; Greter and

Merad, 2013; Rock and Peterson, 2006; Scheiblich and Bicker, 2015a).

Conclusion 31

Conclusion

This thesis describes the interrelationship between NO and CO in regulating cellular

characteristics of microglia under basal conditions and after activation. For reasons

of reducing animal consumption in research the BV-2 cell line was introduced as

model system for primary microglia in functional assays. Our experiments on

microglial activation, cell migration, phagocytosis of neuronal fragment, and the

impact of microglia on neurite outgrowth of human model neurons contribute to better

understand the signaling pathways underlying microglial modulation in pathogenic

conditions (Fig. 9). Moreover, results of the present thesis support a therapeutic

potential of NO synthesis blocking agents and CO-releasing molecules (CORMs) to

medicate excessive inflammation within the CNS and to control the inflammatory

responsiveness of reactive microglia.

Figure 9: Summary of the key findings of the present thesis. Cellular characteristics of microglia

(green), including activation, migration, phagocytosis, and the impact on neurite outgrowth of human

model neurons (magenta), are dually regulated by antagonistic interacting nitric oxide (NO) and

carbon monoxide (CO) signal transduction pathways.

32 References

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Acknowledgements 41

Acknowledgements

There are numerous people who have supported me throughout my doctoral work,

from scientists and colleagues to family and friends.

First and foremost, I thank my supervisor Prof. Dr. Gerd Bicker who offered me the

opportunity to work on this fruitful project, for his unwavering support and guidance

throughout my education, and for his never-ending patience. My time in your

laboratory and the freedom to express my own ideas has transformed me from an

unknowing student to a true and enthusiastic scientist. I really appreciate that you

always believed in my research abilities and taught me to see the bigger picture in

my research. Thank you so much, Gerd!

Secondly, I thank my 2nd evaluator Apl. Prof. Dr. Manuela Gernert who gave me

incredible insights into epilepsy during my master’s course and now kindly agreed to

review my doctoral thesis.

Special thanks are addressed to my co-supervisor PD Dr. Michael Stern who always

had his door and ears open for me. Thank you for very constructive discussions and

helpful suggestions. I always felt welcome and supported by you as you ever had a

hand full of inventiveness! Thank you for teaching me to “think outside the box”!

I thank my co-authors Dr. Vikram Singh and Prof. Dr. Martin Stangel from the

Hannover Medical School who kindly provided me with primary microglia. Thanks to

my colleagues from the Forschergruppe FOR1103 for their contributions regarding

glial culture. Moreover, I really appreciate Prof. Dr. Marcel Leist for the gift of the

BV-2 cell line.

I also thank my former fellow students and the Bicker lab as a whole, but especially

appreciate the support of Dr. Frank Roloff and Saime Tan. Frank, thank you for

teaching me all the needful hinds regarding cell culture. Saime, thank you for always

42 Acknowledgements

having open ears, your technical support, and all the enjoyable coffee breaks during

the last years.

Thanks are also addressed to Prof. Dr. Gerhard Breves who offered me the

opportunity to have my own one-man office and for the shared barbecue activities

during the last years.

Finally, I thank my family and friends without which this work could never have been

performed. My Mom and Dad for always being there for me, and believing in me at

every stage of my life. Thank you for your love, confidence, encouragement, and

never-ending mental support! My sister for always having open ears, and for giving

advice in every condition of life. Thank you for having given birth to my beloved

nephew who really makes me proud! And last, but never ever least, a special big

thank to my friend Anne, who has been a constant strength and steady support not

only in stressful situations but also in our everyday actions. Thank you so much for

teaching me how to deal with pressure and tension, your help to stay focused, your

mental support, and your confidence and love!

THANK YOU!

Eidesstattliche Erklärung 43

Eidesstattliche Erklärung

Hiermit erkläre ich, dass ich die Dissertation „Nitric oxide (NO)- and carbon

monoxide (CO)-mediated signal transduction in a co-culture system of

microglia and human model neurons” selbstständig verfasst habe. Ich habe keine

entgeltliche Hilfe von Vermittlungs- bzw. Beratungsdiensten (Promotionsberater oder

anderer Personen) in Anspruch genommen. Niemand hat von mir unmittelbar oder

mittelbar entgeltliche Leistungen für Arbeiten erhalten, die im Zusammenhang mit

dem Inhalt der vorgelegten Dissertation stehen.

Ich habe die Dissertation an folgenden Institutionen angefertigt:

Stiftung Tierärztliche Hochschule Hannover

Institut für Tierökologie und Zellbiologie, AG Zellbiologie,

Bischofsholer Damm 15/102, 30173 Hannover

Die Dissertation wurde bisher nicht für eine Prüfung oder Promotion oder für einen

ähnlichen Zweck zur Beurteilung eingereicht. Ich versichere, dass ich die

vorstehenden Angaben nach bestem Wissen vollständig und der Wahrheit

entsprechend gemacht habe.

Hannover, den 11. September 2015 ____________________

(Hannah C. Scheiblich)