UUptake and Metabolism of Iron Oxide

Nanoparticles in Cultured Brain Cells

Dissertation

Zur Erlangung eines Doktors in den Naturwissenschaften (Dr. rer. nat.)

Fachbereich 2 (Biologie/Chemie)

Universität Bremen

Charlotte Petters

2015

Erster Gutachter: Professor Dr. Ralf Dringen

Zweiter Gutachter: Professor Dr. Michael Koch

Datum der Verteidigung: 20.02.2015

Hiermit erkläre ich, Charlotte Petters, dass die vorliegende Doktorarbeit selbstständig

und nur unter Verwendung der angegebenen Quellen angefertigt und nicht an anderer

Stelle eingereicht wurde.

Bremen, January 2015

(Charlotte Petters)

TABLE OF CONTENT

I. Acknowledgements ................................................................................................. I

II. Structure of the thesis .......................................................................................... III

III. Summary ............................................................................................................... V

IV. Zusammenfassung .............................................................................................. VII

V. Abbreviations and symbols .................................................................................. IX

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

1.1. Brain cells .............................................................................................................. 3

1.1.1. Neurons ...................................................................................................................... 4

1.1.2. Astrocytes .................................................................................................................... 5

1.1.3. Microglia ..................................................................................................................... 6

1.1.4. Oligodendrocytes ........................................................................................................ 7

1.2. Culture models of brain cells ................................................................................. 8

1.2.1. Neurons ...................................................................................................................... 8

1.2.2. Astrocytes .................................................................................................................. 10

1.2.3. Microglia ................................................................................................................... 10

1.2.4. Oligodendrocytes ...................................................................................................... 11

1.2.5. Co-cultures and slice models.................................................................................... 12

1.3. References ........................................................................................................... 13

1.4. Publication 1 ........................................................................................................ 19

Uptake and metabolism of iron oxide nanoparticles in brain cells

1.5. Publication 2 ........................................................................................................ 23

Handling of iron oxide and silver nanoparticles by astrocytes

1.6. Aim of the thesis .................................................................................................. 27

22. Results .................................................................................................................. 29

2.1. Publication 3 ........................................................................................................ 31

Comparison of primary and secondary rat astrocyte cultures regarding glucose and glutathione metabolism and the accumulation of iron oxide nanoparticles

2.2. Publication 4 ........................................................................................................ 35

Uptake of fluorescent iron oxide nanoparticles by oligodendroglial OLN-93 cells

2.3. Publication 5 ........................................................................................................ 39

Endocytotic uptake of iron oxide nanoparticles by cultured brain microglial cells

2.4. Publication 6 ........................................................................................................ 43

Accumulation of iron oxide nanoparticles by cultured primary neurons

2.5. Publication 7 (manuscript) ................................................................................... 47

Lysosomal iron liberation is responsible for the high vulnerability of brain microglial cells to iron oxide nanoparticles: comparison with neurons and astrocytes

3. Summarizing discussion ....................................................................................... 79

3.1. Synthesis and characterization of fluorescently labeled iron oxide

nanoparticles ........................................................................................................ 81

3.1.1. Characterization of BODIPY-labeled iron oxide nanoparticles ............................ 83

3.1.2. Fluorescent properties of BODIPY-labeled iron oxide nanoparticles .................. 83

3.1.3. Stability of BODIPY-labeled iron oxide nanoparticles .......................................... 84

3.2. Uptake and effects of iron oxide nanoparticles in cultured brain cells ................. 86

3.2.1. Accumulation of iron oxide nanoparticles by cultured brain cells ......................... 87

3.2.2. Uptake of iron oxide nanoparticles in serum-free media ....................................... 90

3.2.3. Uptake of iron oxide nanoparticles in serum-containing media ............................ 91

3.2.4. Cellular localization of iron oxide nanoparticles ..................................................... 92

3.2.5. Toxic effects of an exposure of iron oxide nanoparticles to cultured brain

cells............................................................................................................................ 93

3.3. Implications of the results for the in vivo situation ............................................... 95

33.4. Future perspectives .............................................................................................. 99

3.4.1. Synthesis of fluorescently labeled iron oxide nanoparticles.................................... 99

3.4.2. Mechanism of accumulation of iron oxide nanoparticles under serum-free conditions .................................................................................................................. 99

3.4.3. Binding, uptake and fate of iron oxide nanoparticles in cultured brain cells ...... 100

3.4.4. Effects of an iron oxide nanoparticle exposure to cultured brain cells ............... 101

3.4.5. Investigations on uptake and effects of iron oxide nanoparticles in vivo ............ 102

3.5. References ......................................................................................................... 104

4. Appendix ........................................................................................................... 113

4.1. Curriculum vitae ................................................................................................ 115

4.2. List of publications ............................................................................................ 117

Acknowledgements I

II. ACKNOWLEDGEMENTS At first and particularly, I want to thank Professor Dr. Ralf Dringen. Sincere thanks for

your constant support, help, advice and reliability throughout my thesis which made

working with you so comfortable. Your enthusiasm for science was impressive and I

really appreciate that your door was always open for all kind of questions and that

conversations with you made problems look far less dramatic.

Secondly, I want to thank Professor Dr. Michael Koch for being my second reviewer and

for the nice cooperation while writing the manuscript for the review.

I also want to thank the co-authors of my publications. Thank you for all your

collaboration and valuable comments and discussions. Especially I would like to thank

Dr. Ulf Bickmeyer for the opportunity to use the confocal laser scanning microscope.

Many thanks to all former and recent members of the Dringen working group. I want to

thank all of you for discussions of scientific and non-scientific subjects which made some

stressful days more enjoyable. Maria, it was great sharing the office with you. Thank you

for your open ear and the amusing conversations. Felix, thanks for your great

groundwork on the fluorescent nanoparticles but more importantly for being such a

pleasant and cheerful colleague who always spreads joy. Yvonne, I am grateful to all your

advice in the lab and for the talks which we could squeeze in between. Eva, thank you for

tolerating my visits in your office and for cheering me up. I also want to thank Dr. Maike

Schmidt and Dr. Eva M. Luther for all the fun at the microscope.

Furthermore, I want to thank my family for all the support throughout my life. And last

but not least, I sincerely thank Björn Peter. Thank you for all your encouragements and

mental distraction which made me going on.

Structure of the thesis III

III. STRUCTURE OF THE THESIS The thesis contains the three main chapters introduction, results and summarizing

discussion. The introduction (chapter 1) is composed of two publications and chapters on

brain cells. The results (chapter 2) include four publications and one manuscript. The

publications are embedded as portable network graphics file made from portable

document format files while the manuscript was not accepted at time of submission of the

thesis and hence, is adapted to the style of the thesis. The results are discussed in

summary (chapter 3).

Figures and tables which are not part of a publication are numbered according the

respective main chapter followed by the running number.

Summary V

IIII. SUMMARY Iron oxide nanoparticles (IONPs) are used in various biomedical applications and are

already applied in human therapy. Since IONPs can reach the brain, detailed knowledge

on uptake and effects of IONPs on neural cells is required. In the present thesis, IONPs

were synthesized and fluorescently labeled by attaching the green fluorescent dye

BODIPY to the coating material dimercaptosuccinate. When 1.5% of the thiol groups of

dimercaptosuccinate were functionalized, IONPs had identical physicochemical

properties to the non-fluorescent version of the IONPs and were therefore considered as

suitable fluorescent tool to study uptake and intracellular localization of

dimercaptosuccinate-coated IONPs.

Cellular uptake, localization and potential toxic effects of IONPs were investigated in cell

culture models of the four major neural cell types (neurons, astrocytes, microglia,

oligodendrocytes). In general, neural cell cultures efficiently accumulated IONPs which

led to an increase of the specific cellular iron contents to maximal levels of up to 3000

nmol iron per mg protein. The uptake of IONPs in cultured brain cells strongly

depended on experimental conditions such as time of incubation, IONP concentration,

temperature and on the absence or presence of serum. In the presence of serum, the

accumulation of IONPs was decreased by 80-90% in all cell types investigated compared

to serum-free conditions. Dependent on the cell type investigated, IONP uptake in

presence of serum was strongly lowered by known inhibitors of endocytotic processes

suggesting involvement of clathrin-mediated endocytosis and/or macropinocytosis. In

contrast, for IONP uptake in absence of serum the pathways involved remain to be

elucidated.

A direct comparison of cultured astrocytes, neurons and microglia revealed that microglia

were most efficient in IONP accumulation but also highly vulnerable to IONP exposure.

Microglial cell death was prevented by neutralizing lysosomes or by chelating iron ions,

suggesting that toxicity is mediated by rapid transfer of IONPs to lysosomes and fast

IONP degradation in the acidic environment which resulted in microglial death by iron-

mediated oxidative stress. In contrast to microglia, primary astrocytes, neurons and

oligodendroglial OLN-93 cells were not acutely damaged within hours upon IONP

exposure. However, at least neurons which had accumulated substantial amounts of

VI Summary

IONPs during a short time exposure suffered from delayed toxicity after removal of

exogenous IONPs.

The data presented in this thesis reveal that brain cells deal well with low amounts of

IONPs. However, higher iron contents after IONP exposure cause acute or delayed

toxicity in some neural cell types. Among the different cell types, especially microglia

were vulnerable to IONPs. Hence, concerning biomedical application of IONPs to the

brain one should consider protecting microglia from IONP-derived stress to reduce or

prevent potential adverse effects to the brain.

Zusammenfassung VII

IIV. ZUSAMMENFASSUNG Eisenoxidnanopartikel (IONPs) finden eine breite Anwendung in der Biomedizin und

werden bereits für Therapien eingesetzt. Da IONPs das Gehirn erreichen können, ist

eine detaillierte Kenntnis über ihre Aufnahme in und Wirkung auf Gehirnzellen wichtig.

In der vorliegenden Arbeit wurden IONPs synthetisiert und fluoreszenzmarkiert. Dazu

wurde der grün-fluoreszierende Farbstoff BODIPY an das Hüllmaterial

Dimercaptobernsteinsäure angehängt. Die Menge an BODIPY in der Hülle hat einen

großen Einfluss auf die Stabilität. Nach Markierung von 1.5% der Thiolgruppen der

Dimercaptobernsteinsäure in der Hülle hatten diese IONPs identische physikochemische

Eigenschaften wie die nicht-markierten IONPs. Daher sind BODIPY-markierte IONPs

geeignet um die intrazelluläre Lokalisierung von Dimercaptobernsteinsäure-ummantelten

IONPs zu untersuchen.

Zelluläre Aufnahme, Lokalisierung und mögliche toxischen Effekte von IONPs wurden

in Zellkulturmodellen der vier Hauptzelltypen des Gehirns (Neurone, Astrozyten,

Microglia, Oligodendrozyten) untersucht. Im Allgemeinen akkumulierten neurale

Zellkulturen IONPs effizient, was zu einem Anstieg ihres spezifischen zellulären

Eisengehalts auf maximale Werte von bis zu 3000 nmol Eisen pro mg Protein führte. Die

IONP-Aufnahme in Gehirnzellkulturen hing stark von den experimentellen Bedingungen

wie Inkubationszeit, verwendete IONP-Konzentration, Temperatur und von der An-

oder Abwesenheit von Serum ab. In der Anwesenheit von Serum nahm die

Akkumulierung von IONPs im Vergleich zu serumfreien Bedingungen in allen

untersuchten Zellkulturen um 80-90% ab. Die IONP-Aufnahme in Anwesenheit von

Serum wurde durch bekannte Inhibitoren für endozytotische Wege stark verringert. Dies

weist darauf hin, dass abhängig vom Zelltyp Clathrin-vermittelte Endozytose und/oder

Macropinozytose an der IONP-Aufnahme in Gehirnzellen beteiligt sind. Im Gegensatz

dazu müssen die für die IONP-Aufnahme verantwortlichen Wege in Abwesenheit von

Serum noch aufgeklärt werden.

Ein direkter Vergleich von kultivierten Astrozyten, Neuronen und Microglia zeigte, dass

Microglia am effizientesten IONPs akkumulierten, aber auch besonders anfällig für

IONP-vermittelte Toxizität waren. Ihr Zelltod konnte durch eine Neutralisierung der

Lysosomen oder durch Chelatierung von Eisenionen verhindert werden. Dies lässt darauf

schließen, dass die Toxizität durch schnellen Transport der IONPs zu Lysosomen und

VIII Zusammenfassung

schnellen IONP-Auflösung im sauren Milieu, was zu eisenvermittelten oxidativen Stress

führt. Im Gegensatz zu Microglia wurden primäre Astrozyten, Neurone oder

oligodendrogliale OLN-93-Zellen durch IONP-Exposition nicht akut innerhalb von

wenigen Stunden geschädigt. Allerdings zeigten zumindest Neurone, welche substanzielle

Mengen IONPs während einer kurzen Inkubationszeit akkumulierten, eine verzögerte

Toxizität nach dem Entfernen von exogenen IONPs.

Die Daten der vorliegenden Arbeit zeigen, dass Gehirnzellen gut mit geringen Mengen an

IONPs umgehen können. Höhere Eisengehalte nach IONP-Behandlung können jedoch

zu akuter oder verzögerter Toxizität führen. Unter den verschiedenen Gehirnzelltypen

waren besonders Microglia anfällig für IONP-vermittelten Stress. Daher sollte für

biomedizinische Anwendungen von IONPs ein Schutz der Microglia in Betracht gezogen

werden um mögliche Schädigungen im Gehirn zu verhindern oder zu verringern.

Abbreviations and symbols IX

VV. ABBREVIATIONS AND SYMBOLS °C degree Celsius % percent ζ zeta ADP adenosine diphosphate ANOVA analysis of variance APCs astroglia-rich primary cultures ASCs astroglia-rich secondary cultures ATP adenosine triphosphate ATPase adenosine triphosphatase Baf1 Bafilomycin A1 BBB blood-brain barrier bipy bipyridyl BP, BODIPY (FL C1-IA) N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-

3-yl)methyl)iodoacetamide BP-(D)-IONPs BP-labeled DMSA-coated IONPs CME clathrin-mediated endocytosis CNS central nervous system CS citrate synthase CvME caveolin-mediated endocytosis DAPI 4’,6-diamidino-2-phenylindole D-IONPs dimercaptosuccinate-coated IONPs DMEM Dulbecco’s modified Eagles medium DMSA dimercaptosuccinic acid DMT1 divalent metal transporter 1 Eds. editors EDX energy dispersive X-ray spectroscopy e.g. for example (Latin: exempli gratia) EIPA 5-(N-ethyl-N-isopropyl)amiloride etc. and so on (Latin: et cetera) FCS fetal calf serum FT-IR Fourier transform infrared spectroscopy G6PDH glucose-6-phosphate dehydrogenase GAPDH glyceraldehyde-3-phosphate dehydrogenase GCM glia-conditioned medium GFAP glial fibrillary acidic protein GR glutathione reductase GS glutamine synthetase GSH glutathione GSSG glutathione disulfide GSx total glutathione (GSH + 2 GSSG) GTPase guanosine triphosphatase

X Abbreviations and symbols

HEPES 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid HO-1 heme oxygenase-1 IB incubation buffer i.e. that is (Latin: id est) IgG immunoglobulin G IL interleukin IONPs iron oxide nanoparticles LDH lactate dehydrogenase LT lysotracker MAG myelin-associated glycoprotein MEM minimal essential media MFH magnetic fluid hyperthermia MP macropinocytosis MRI magnetic resonance imaging MT metallothionein MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-

(4-sulfophenyl)-2H-tetrazolium MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide NAD(P)H nicotinamide adenine dinucleotide (phosphate) NPs nanoparticles P phagocytosis PBS phosphate-buffered saline PEG polyethylene glycol PVP polyvinylphenol PI propidium iodide ppm parts per million Rab Ras (Rat sarcoma) related in brain RME receptor mediated endocytosis RNA ribonucleic acid ROS reactive oxygen species RT room temperature SD standard deviation T temperature TEM transmission electron microscopy TNF-α tumor necrosis factor-α w/v weight per volume

1. IIntroduction

1.1. Brain cells .............................................................................................................. 3 1.1.1. Neurons ...................................................................................................................... 4

1.1.2. Astrocytes .................................................................................................................... 5

1.1.3. Microglia ..................................................................................................................... 6

1.1.4. Oligodendrocytes ........................................................................................................ 7

1.2. Culture models of brain cells ................................................................................. 8 1.2.1. Neurons ...................................................................................................................... 8

1.2.2. Astrocytes .................................................................................................................. 10

1.2.3. Microglia ................................................................................................................... 10

1.2.4. Oligodendrocytes ...................................................................................................... 11

1.2.5. Co-cultures and slice models.................................................................................... 12

1.3. References ........................................................................................................... 13

1.4. Publication 1 ........................................................................................................ 19 Petters C, Irrsack E, Koch M, Dringen R (2014) Uptake and metabolism of iron oxide nanoparticles in brain cells. Neurochem Res 39:1648-1660

1.5. Publication 2 ........................................................................................................ 23 Hohnholt MC, Geppert M, Luther EM, Petters C, Bulcke F, Dringen R (2013) Handling of iron oxide and silver nanoparticles by astrocytes. Neurochem Res 38:227-239

1.6. Aim of the thesis .................................................................................................. 27

Introduction 3

11. Introduction

1.1. Brain cells

Brain cells can be divided into neurons and glial cells (Peters and Connor, 2014; Figure

1). The glial cells are subdivided into astrocytes, oligodendrocytes and microglia. The

ratio of glia to neurons in the human brain is a matter of debate. While some reviews

state that glia, especially astrocytes, outnumber neurons by 10-fold without mentioning the

source (Allen and Barres, 2009; Sofroniew and Vinters, 2010; Verkhratsky and Parpura,

2010), other reports claim that the number of neuronal and non-neuronal cells are almost

equal with around 86 billion neurons versus 84 billion non-neuronal cells (Azevedo et al.,

2009; Herculano-Houzel, 2014). However, the glia/neuron ratio is quite diverse in

different parts of the brain such as cerebellum or cerebral cortex (Herculano-Houzel,

2014).

The following chapters will describe the physiology and function of neurons, astrocytes,

microglia and oligodendrocytes and the commonly used cell culture models of these cell

types.

Figure 1.1 The four major cell types in the brain: neurons (yellow), oligodendrocytes (blue), astrocytes (green) covering a blood vessel (red), microglia (violet).

4 Introduction

11.1.1. Neurons

Neurons were firstly described by Jan Evangelista Purkinje in 1839 (Raine, 2006) and

make up around 50% of the human brain (Azevedo et al., 2009; Herculano-Houzel,

2014). They are composed of a perikaryon or soma, a varying number of branched

dendrites and one axon which can be branched, thereby forming many synapses (Raine,

2006). The neuronal somata are located in the grey matter whereas the white matter

contains the axons (Raine, 2006). In general, neurons have the distinct function to

transmit electrochemical signals. The action potential is built at the axon hillock by the all-

or-none-law by the influx of sodium ions through voltage gated sodium channels, resulting

in a depolarization of the plasma membrane (Hille and Catterall, 2006). The signal is

transferred along the axon to the axon terminal where neurotransmitters stored in vesicles

are released by exocytosis into the synaptic cleft (Scalettar, 2006; Stevens and Williams,

2000). Subsequently, neurotransmitters bind to receptors at the postsynaptic membrane

of neighboring neurons and are cleaved (Chang et al., 1985; Silman and Sussman, 2005)

or taken up to prevent permanent excitation (Balcar and Johnston, 1972; Palmer et al.,

2003; Kondratskaya et al., 2010). The binding of the neurotransmitter to the receptor

leads to an either excitatory or inhibitory response of the postsynaptic neuron, thereby

facilitating or hinder formation of action potentials, respectively (Holz and Fisher, 2006;

Peters and Connor, 2014). The signal transduction is a highly energy consuming process

but the axon can be very elongated (up to 1 m). Hence, there are complex trafficking

mechanisms to transport mitochondria retrograde or anterograde along the axon (Sheng,

2014).

Neurons can differ regarding their morphology, location, function (motor, sensory) or

effect (excitatory, inhibitory) (Holz and Fisher, 2006; Guerout et al., 2014; Pasca et al.,

2014). For example, cerebellar granule neurons possess a small soma (6-8 μm) (Monteiro

et al., 1998; Raine, 2006) while the diameter of the soma of Purkinje cells is about 30 μm

(Floeter and Greenough, 1979; Takacs and Hamori, 1994). Beside the size, the number

and complexity of processes can differ extensively (Raine, 2006). Moreover, neurons can

specialize to sensory neurons possessing specific receptors which are activated by a certain

stimulus (Holz and Fisher, 2006) or to motor neurons which innervate muscles und

stimulate muscle cells via the neuronal end plate (Grumbles et al., 2012; Tanaka et al.,

2014). The effect of neurotransmitters depends on the receptor in the postsynaptic

membrane. Herein, one neurotransmitter can either excite or inhibit the postsynaptic

neuron (Holz and Fisher, 2006).

Introduction 5

11.1.2. Astrocytes

In the beginning of glial research, astrocytes were thought to give only structural support.

In the meanwhile, a variety of important astrocytic functions was discovered (Sofroniew

and Vinters, 2010) and due to their morphological and physiological heterogeneity it is

quite difficult to define what an astrocyte is (Kettenmann and Verkhratsky, 2011).

Astrocytes are named by their often star-like morphology (Oberheim et al., 2012). The

astrocytic end-feet cover the majority of the blood-brain barrier (BBB) which makes them

the first neural cell type to get in contact with compounds passing the BBB (Virgintino et

al., 1997; De Bock et al., 2014). They not only provide other neural cells with energy

substrates, they also play a key role in the homeostasis of ions, pH and water (Kimelberg,

2010; Sofroniew and Vinters, 2010) and they regulate the vasopressure in brain (De Bock

et al., 2014; Howarth, 2014). Moreover, they interact with neurons and modulate their

signal transmission. Considering the “tripartite synapse hypothesis”, the synapse is

composed of the axonal terminal, the synaptic cleft, the postsynaptic membrane and

additionally astrocytes in close proximity to the synapse. Due to expression of

transporters astrocytes can actively take up neurotransmitters released from the synaptic

cleft (Kettenmann and Verkhratsky, 2011; Roberts et al., 2014; Verkhratsky et al., 2014)

to prevent neurons from excitotoxicity (Oberheim et al., 2012) or they can release

gliotransmitters such as ATP, glutamate, serine or γ-amino butyric acid to modulate

signaling (Verkhratsky and Parpura, 2010; Zhuang et al., 2011; Lee et al., 2013; De Bock

et al., 2014). Astrocytes communicate with each other via intercellular calcium waves

through gap junctions and hence, can modulate synaptic activity of distinct synapses (De

Bock et al., 2014; Lallouette et al., 2014).

Due to their location, astrocytes are the first neural cell type which comes into contact

with xenobiotics crossing the BBB. Thus, they have also defense systems to metabolize

and export xenobiotics (Dringen et al., 2015) and to protect neurons from stress (Wilson

et al., 2000; Barreto et al., 2011; Genis et al., 2014). In case of cerebral injury, a glial scar

is formed by reactive astrocytes which are characterized by strong expression of glial

fibrillary acidic protein (GFAP) (Cregg et al., 2014; Liu et al., 2014) which is used as

specific astrocytic marker although not all astrocytes express GFAP (Kettenmann and

Verkhratsky, 2011).

6 Introduction

11.1.3. Microglia

The brain is described as immune privileged which means immune competent cells from

the blood are excluded from the central nervous system (CNS) (Kettenmann et al., 2011).

During early development mesodermal precursor cells enter the brain and differentiate

into microglia which are macrophage-like cells and represent the immune competent cells

of the CNS (Neumann and Wekerle, 2013; Nayak et al., 2014; Peters and Connor,

2014). Due to their phagocytic properties, they incorporate pathogens (bacteria, viruses,

fungi, parasites) invading the brain or they take up cell debris in case of apoptosis/necrosis

of neural cells (Eyo and Dailey, 2013; Nayak et al., 2014). In the healthy CNS, microglia

are “resting” and possess a ramified morphology while under pathological conditions they

get activated and turn into an amoeboid shape which was firstly described by Pío del Río-

Hortega (Wilms et al., 1997; Biber et al., 2014). This activation might be induced by

binding of certain compounds such as purines, released from injured neurons or glia, to

specific receptors in the microglial membrane (Eyo and Dailey, 2013; Bernier et al.,

2013). In the activated state, microglia secrete anti- and pro-inflammatory cytokines such

as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) or nitric oxide (Kettenmann et

al., 2011; Matsui et al., 2014; Pisanu et al., 2014).

An important feature of microglia is their mobility, including migration of the whole cells

and motility of the processes, thereby scanning their environment (Eyo and Dailey, 2013;

Nayak et al., 2014). Microglia make 5% of glial cells in the grey matter (Pelvig et al., 2008)

and they are in general regularly distributed within the brain possessing certain territories

(Milligan et al., 1991; Neumann and Wekerle, 2013). However, if microglia become

activated they guide surrounding microglia to the site of interference and start to

proliferate to increase the number of microglia at the damaged location (Nayak et al.,

2014; Marlatt et al., 2014). Besides their neuroprotective nature, microglia play also a role

during brain development, supporting the angiogenesis and controlling neuronal

apoptosis and synaptic homeostasis (Eyo and Dailey, 2013; Nayak et al., 2014).

Moreover, the activated state of microglia is discussed to be neurotoxic and associated

with neurodegeneration and aging of the brain, though this is still a matter of debate

(Biber et al., 2014).

Introduction 7

11.1.4. Oligodendrocytes

Oligodendrocytes are the most abundant glial cell type in the cerebral cortex (Pelvig et al.,

2008) and derive from oligodendrocyte precursor cells (Guerout et al., 2014; Pedraza et

al., 2014). The differentiation of these precursor cells into mature oligodendrocytes is

complex, involving loss of migration and proliferation and formation of processes (Bauer

et al., 2009; Bradl and Lassmann, 2010). The unique function of mature

oligodendrocytes is the myelination of neuronal axons by enwrapping the axons with

several layers of membrane (Derobertis et al., 1958; Aggarwal et al., 2011). A single

oligodendrocyte can ensheath 50 axons (Guerout et al., 2014) and completes its myelin

sheaths within few days (Watkins et al., 2008). The production of the myelin which is

composed of more than 70% lipids of the dry weight (Norton and Poduslo, 1973;

Aggarwal et al., 2011) consumes a lot of energy and oxygen which results in generation of

reactive oxygen species (ROS) (Bradl and Lassmann, 2010). As enzymes involved in

myelination depend on iron as cofactor, oligodendrocytes are the neural cells with the

highest iron content which makes oligodendrocytes vulnerable to oxidative stress caused

by elevated ROS production via the Fenton reaction (Thorburne and Juurlink, 1996;

Bradl and Lassmann, 2010).

The myelin sheath is interrupted in regular intervals of 100 μm (Kettenmann and

Verkhratsky, 2011), leaving the axon uncovered. These areas are called nodes of Ranvier.

Since the myelin sheaths electrically isolate the axon, no action potential can form but at

the nodes of Ranvier. This leads to saltatory signal conduction (Tasaki, 1939; Bradl and

Lassmann, 2010) and to a dramatic increase in the speed which can otherwise only be

achieved with very large unmyelinated axons with a diameter of 1 mm as in the giant

squid (Kettenmann and Verkhratsky, 2011). Besides the isolation of the axons, myelin

sheaths also influence neuronal structure and physiology such as axon diameter and

transport rates along the axon (Bradl and Lassmann, 2010) and provide the axon with

energy substrates such as lactate which is crucial for axonal health (Lee et al., 2012;

Funfschilling et al., 2012; Morrison et al., 2013; Rinholm and Bergersen, 2013).

8 Introduction

1.2. CCulture models of brain cells

Studying the metabolism of neural cells or effects of xenobiotics on brain is crucial to

understand the function and pathology of the brain but is simultaneously complicated due

to the complexity of the brain. Here, cell culture systems provide a convenient tool to

investigate a variety of metabolic parameters in the cell type of interest also from certain

brain regions under controlled culture conditions without the need of animal experiments

(Hansson and Thorlin, 1999; Lange et al., 2012). Yet, in vitro studies lack of the three

dimensional organization of the tissue, of cell cell interactions mainly between different

cell types and the environment within the living organism (Hansson and Thorlin, 1999).

Additionally, these systems are quite artificial such as culture medium which often has a

completely different composition than the cerebrospinal fluid. This divergence from the

in vivo situation has to be considered when interpreting the results. Nevertheless,

analyzing the effect of compounds on brain cells in culture give a hint to what might

happen in vivo (Sunol et al., 2008) and a lot of knowledge about brain cells was obtained

by cultured neural cells (Lange et al., 2012; Tulpule et al., 2014).

Most of brain cell cultures are obtained from rodents. There are diverse model systems

for the different brain cells, most of them are primary (deriving directly from brain tissue)

or secondary cultures (reseeded cells obtained from primary cultures). However, some

research is also performed on cell lines which have some advantages such as no need of

animals, easy culture techniques and nearly unlimited supply of cells due to cell division.

On the other hand, cell lines are immortalized, having often cancerous origin which might

imply an altered metabolism and different response to stimuli. Due to the different

culturing techniques (tissue derived cells, cell lines, days in vitro, medium composition,

coating of the plates etc.) it is important to use a cell culture system which is appropriate

for the question or purpose (Lange et al., 2012).

1.2.1. Neurons

Primary neuron cultures are mainly obtained from distinct regions of the brain (e.g.

hippocampus, cerebellum or cortex) rather than from the whole brain and might be

either prepared from embryonal or from 7 d old rodents which depends on the brain

region due to varying differentiation states (Sunol et al., 2008). Cultures of cortical and

cerebellar granule neurons have been shown to not differ from each other with respect to

respiration (Jameson et al., 1984) or chloride uptake (Sunol et al., 2008). In the present

Introduction 9

thesis, cultures of cerebellar granule neurons (Figure 2a) were used. They express

neuronal markers such as microtubule-associated protein 2 (Tulpule et al., 2014). Like

the majority of neurons in vivo, cultured primary neurons do not divide after seeding but

elongate their processes. As the yield of preparation of primary neuron cultures is

relatively low (Sunol et al., 2008), many research groups use the cell line PC12 or to a

lesser extent SH-SY5Y instead. They have rat phaeochromocytoma (Westerink and

Ewing, 2008) and human neuroblastoma (Xie et al., 2010) as origin, respectively and are

often used as a model for differentiating neurons but also to study metabolism and

toxicity in neurons (Xie et al., 2010; Westerink and Ewing, 2008).

FFigure 2.1 Cell cultures of brain cells used in the present thesis: primary cerebellar granule neurons (a), OLN-93 cells (b), astroglia-rich primary culture (c; from chapter 2.1) and primary microglial cells (d). Pictures were captured using an Eclipse TE-2000U (a, b, c) or TS-100 (d) microscope from Nikon (Düsseldorf, Germany). The scale bars represent 100 μm.

10 Introduction

11.2.2. Astrocytes

Astrocytes are frequently used as primary or secondary culture (Lange et al., 2012) while

C6 glioma cells are used a model for astroglioma cells (Barth and Kaur, 2009). Astroglia-

rich primary cultures (Figure 2b) are prepared from neonatal rodents (Hamprecht and

Löffler, 1985) and are contaminated by other glial cells (chapter 2.1; Saura, 2007; Yoo

and Wrathall, 2007). The heterogeneity of astrocytes within the living brain is challenging

for culturing astrocytes and interpreting the experimental outcome (Lange et al., 2012).

Already astroglia-rich primary cultures prepared from rats and mice respond differently

under the same culture conditions (Ahlemeyer et al., 2013). Cultured astrocytes possess

the same properties as astrocytes in brain such as glycogen formation (Dringen et al.,

1993), glutamate transport (Hertz et al., 1978) or storage of metal ions (Jones et al., 2012).

While astrocytes in vivo express the astrocytic marker GFAP only in activated state (Cregg

et al., 2014), cultured astrocytes are mainly GFAP-positive (Du et al., 2010). However,

this depends on culturing conditions (chapter 2.1; Goetschy et al., 1986; Codeluppi et al.,

2011). To explore astrocyte behavior in diseases, different in vitro models are used such

as scratch injury for trauma or oxygen glucose deprivation for ischemia (Yu et al., 1993;

Wu and Schwartz, 1998; Giffard and Swanson, 2005; Lange et al., 2012).

1.2.3. Microglia

There are two major preparation techniques described to obtain microglial cultures.

Mixed glial cultures contain besides astrocytes a certain amount of other glial cell such as

oligodendrocytes, ependymal cells and microglia and these contaminating cells are either

sitting on top of the astrocyte layer or underneath it (Saura et al., 2003; Yoo and Wrathall,

2007). Hence, microglia can be isolated by shaking the culture for a certain time to detach

the top layer microglia and subsequently, the microglia-containing medium is removed

and used for seeding into new culture plates (Tamashiro et al., 2012; Deierborg, 2013). In

the other method, the astrocytic layer is detached by trypsination and removed while the

bottom layer microglia remain in the plate (Saura et al., 2003; Figure 2d). As their in vivo

counterparts, cultured microglia are mobile (Jeon et al., 2012), express proteins for

immune response such as CD11b (Szabo and Gulya, 2013) and release nitric oxide if they

become activated (Saura et al., 2003). However, isolated microglia display in general

amoeboid morphology as seen for activated microglia in vivo while microglia in mixed

glial cultures are more ramified (Saura et al., 2003). Besides the described primary and

Introduction 11

secondary microglial cultures, cell lines such as BV2 or N9 which are derived from

murine microglia and immortalized by viruses are used as microglial model systems

(Stansley et al., 2012; Rodhe, 2013). These cell lines have the advantage that they

overcome the low yield problem of the primary cultures while possessing the same

microglial characteristics. Nevertheless, unlimited cell division might alter differentiation

and properties (Rodhe, 2013).

11.2.4. Oligodendrocytes

Cultured oligodendrocytes are obtained similarly to microglia by shaking off top layer

cells from astroglia-rich primary cultures (McCarthy and de Vellis, 1980; Barateiro and

Fernandes, 2014). While microglia are more easily detaching than oligodendrocytes,

there are often two shaking steps used to lower microglial contamination in the

oligodendrocyte culture (Barateiro and Fernandes, 2014). As observed for

oligodendroglial cells in vivo, also in culture these cells can be divided into

oligodendrocyte progenitor cells, pre-oligodendrocytes, immature and mature

oligodendrocytes, as characterized by morphology and expression of specific proteins

(Jarjour et al., 2012; Barateiro and Fernandes, 2014). Oligodendrocytic differentiation can

be provoked by e.g. thyroid hormone (Barateiro et al., 2013) or by the presence of axons

in co-cultures (Barateiro and Fernandes, 2014). Compared to oligodendrocytes in vivo,

cultured oligodendrocytes produce 500-times less area of myelin membrane (Jarjour et

al., 2012). Oligodendrocyte culturing methods are limited by a very low cell yield and the

fact that mature oligodendrocytes do not divide in vitro (Buntinx et al., 2003). Also here,

cell lines are helpful. Herein, human clonal cell lines such as HOG, MO3.13 and KG-1C

(Buntinx et al., 2003) as well as the rat cell line OLN-93 (Richter-Landsberg and

Heinrich, 1996) are available. Advantageously, OLN-93 cells (Figure 2b) are not derived

from a tumor as HOG and KG-1C but they are spontaneously transformed cells from

primary glial cultures (Richter-Landsberg and Heinrich, 1996). OLN-93 cells possess

markers for differentiated oligodendroglial cells such as galactocerebroside, myelin-

associated glycoprotein or myelin-basic protein though they resemble morphologically

more oligodendrocyte progenitors and are able to proliferate (Richter-Landsberg and

Heinrich, 1996).

12 Introduction

11.2.5. Co-cultures and slice models

Besides the conventional cell culture techniques described above there is emphasis to

mimic the in vivo situation more realistically. To allow and investigate interactions

between different neural cell types, co-cultures can be prepared (Jones et al., 2012;

Welser and Milner, 2012; Jarjour et al., 2012). This type of cell culture was very useful to

examine different aspects of interactions between cell types. For example, the release of

glutathione and extracellular degradation by astrocytes supplies neurons with the

precursors to synthesize glutathione intracellularly (Dringen et al., 1999). While

cerebellar granule neurons in vitro express glutamine synthetase, an enzyme in vivo solely

expressed in astrocytes (Norenberg and Martinez-Hernandez, 1979; Albrecht et al.,

2007), when deprived in glutamine, co-culturing with astrocytes reduced or abolished

glutamine synthetase expression in neurons (Fernandes et al., 2010). Moreover, the

formation of myelin by oligodendrocytes is enhanced by presence of astrocytes (Watkins

et al., 2008).

Additionally, ex vivo cultures of brain slices of around 200-500 μm thickness preserve the

three dimensional organization of the brain and have been successfully used to identify

the myelinating cell type in the CNS (Jarjour et al., 2012). To overcome the two

dimensional culture model, there are approaches to culture brain cells three

dimensionally on scaffolds (Kang et al., 2014; Lau et al., 2014; Weightman et al., 2014) or

as neurospheres which are free floating aggregates of neural stem cells (Brito et al., 2012;

Ghate et al., 2014). However, there will always be the drawback that human brain cell

cultures are rare and the conclusions based on results from rodent cell cultures to human

brain are hard to draw.

Introduction 13

1.3. RReferences

Aggarwal, S., Yurlova, L. & Simons, M. (2011). Central nervous system myelin: structure, synthesis and assembly. Trends Cell Biol 21: 585-593.

Ahlemeyer, B., Kehr, K., Richter, E., Hirz, M., Baumgart-Vogt, E. & Herden, C. (2013). Phenotype, differentiation, and function differ in rat and mouse neocortical astrocytes cultured under the same conditions. J Neurosci Methods 212: 156-164.

Albrecht, J., Sonnewald, U., Waagepetersen, H. S. & Schousboe, A. (2007). Glutamine in the central nervous system: function and dysfunction. Front Biosci 12: 332-343.

Allen, N. J. & Barres, B. A. (2009). Neuroscience: glia - more than just brain glue. Nature 457: 675-677.

Azevedo, F. A., Carvalho, L. R., Grinberg, L. T., Farfel, J. M., Ferretti, R. E., Leite, R. E., Jacob Filho, W., Lent, R. & Herculano-Houzel, S. (2009). Equal numbers of neuronal and nonneuronal cells make the human brain an isometrically scaled-up primate brain. J Comp Neurol 513: 532-541.

Balcar, V. J. & Johnston, G. A. (1972). The structural specificity of the high affinity uptake of L-glutamate and L-aspartate by rat brain slices. J Neurochem 19: 2657-2666.

Barateiro, A. & Fernandes, A. (2014). Temporal oligodendrocyte lineage progression: in vitro models of proliferation, differentiation and myelination. Biochim Biophys Acta 1843: 1917-1929.

Barateiro, A., Miron, V. E., Santos, S. D., Relvas, J. B., Fernandes, A., Ffrench-Constant, C. & Brites, D. (2013). Unconjugated bilirubin restricts oligodendrocyte differentiation and axonal myelination. Mol Neurobiol 47: 632-644.

Barreto, G. E., Gonzalez, J., Torres, Y. & Morales, L. (2011). Astrocytic-neuronal crosstalk: implications for neuroprotection from brain injury. Neurosci Res 71: 107-113.

Barth, R. F. & Kaur, B. (2009). Rat brain tumor models in experimental neuro-oncology: the C6, 9L, T9, RG2, F98, BT4C, RT-2 and CNS-1 gliomas. J Neurooncol 94: 299-312.

Bauer, N. G., Richter-Landsberg, C. & Ffrench-Constant, C. (2009). Role of the oligodendroglial cytoskeleton in differentiation and myelination. Glia 57: 1691-1705.

Bernier, L. P., Ase, A. R., Boue-Grabot, E. & Seguela, P. (2013). Inhibition of P2X4 function by P2Y6 UDP receptors in microglia. Glia 61: 2038-2049.

Biber, K., Owens, T. & Boddeke, E. (2014). What is microglia neurotoxicity (not)? Glia 62: 841-854.

Bradl, M. & Lassmann, H. (2010). Oligodendrocytes: biology and pathology. Acta Neuropathol 119: 37-53.

Brito, C., Simao, D., Costa, I., Malpique, R., Pereira, C. I., Fernandes, P., Serra, M., Schwarz, S. C., Schwarz, J., Kremer, E. J. & Alves, P. M. (2012). Generation and genetic modification of 3D cultures of human dopaminergic neurons derived from neural progenitor cells. Methods 56: 452-460.

Buntinx, M., Vanderlocht, J., Hellings, N., Vandenabeele, F., Lambrichts, I., Raus, J., Ameloot, M., Stinissen, P. & Steels, P. (2003). Characterization of three human oligodendroglial cell lines as a model to study oligodendrocyte injury: morphology and oligodendrocyte-specific gene expression. J Neurocytol 32: 25-38.

Chang, C. C., Hong, S. J., Lin, H. L. & Su, M. J. (1985). Acetylcholine hydrolysis during neuromuscular transmission in the synaptic cleft of skeletal muscle of mouse and chick. Neuropharmacology 24: 533-539.

14 Introduction

Codeluppi, S., Gregory, E. N., Kjell, J., Wigerblad, G., Olson, L. & Svensson, C. I. (2011). Influence of rat substrain and growth conditions on the characteristics of primary cultures of adult rat spinal cord astrocytes. J Neurosci Methods 197: 118-127.

Contestabile, A. (2002). Cerebellar granule cells as a model to study mechanisms of neuronal apoptosis or survival in vivo and in vitro. Cerebellum 1: 41-55.

Cregg, J. M., DePaul, M. A., Filous, A. R., Lang, B. T., Tran, A. & Silver, J. (2014). Functional regeneration beyond the glial scar. Exp Neurol 253: 197-207.

De Bock, M., Decrock, E., Wang, N., Bol, M., Vinken, M., Bultynck, G. & Leybaert, L. (2014). The dual face of connexin-based astroglial Ca2+ communication: a key player in brain physiology and a prime target in pathology. Biochim Biophys Acta 1843: 2211-2232.

Deierborg, T. (2013). Preparation of primary microglia cultures from postnatal mouse and rat brains. Methods Mol Biol 1041: 25-31.

Derobertis, E., Gerschenfeld, H. M. & Wald, F. (1958). Cellular mechanism of myelination in the central nervous system. J Biophys Biochem Cyt 4: 651-656.

Dringen, R., Gebhardt, R. & Hamprecht, B. (1993). Glycogen in astrocytes: possible function as lactate supply for neighboring cells. Brain Res 623: 208-214.

Dringen, R., Pfeiffer, B. & Hamprecht, B. (1999). Synthesis of the antioxidant glutathione in neurons: supply by astrocytes of CysGly as precursor for neuronal glutathione. J Neurosci 19: 562-569.

Dringen, R., Brandmann, M., Hohnholt, M. C. & Blumrich, E. M. (2015). Glutathione-dependent detoxification processes in astrocytes. Neurochem Res, in press.

Du, F., Qian, Z. M., Zhu, L., Wu, X. M., Qian, C., Chan, R. & Ke, Y. (2010). Purity, cell viability, expression of GFAP and bystin in astrocytes cultured by different procedures. J Cell Biochem 109: 30-37.

Eyo, U. B. & Dailey, M. E. (2013). Microglia: key elements in neural development, plasticity, and pathology. J Neuroimmune Pharmacol 8: 494-509.

Fernandes, S. P., Dringen, R., Lawen, A. & Robinson, S. R. (2010). Neurones express glutamine synthetase when deprived of glutamine or interaction with astrocytes. J Neurochem 114: 1527-1536.

Floeter, M. K. & Greenough, W. T. (1979). Cerebellar plasticity: modification of Purkinje cell structure by differential rearing in monkeys. Science 206: 227-229.

Funfschilling, U., Supplie, L. M., Mahad, D., Boretius, S., Saab, A. S., Edgar, J., Brinkmann, B. G., Kassmann, C. M., Tzvetanova, I., D., Möbius, W., Diaz, F., Meijer, D., Suter, U., Hamprecht, B., Sereda, M. W., Moraes, C. T., Frahm, J., Goebbels, S. & Nave, K. A. (2012). Glycolytic oligodendrocytes maintain myelin and long-term axonal integrity. Nature 485: 517-521.

Genis, L., Davila, D., Fernandez, S., Pozo-Rodrigalvarez, A., Martinez-Murillo, R. & Torres-Aleman, I. (2014). Astrocytes require insulin-like growth factor I to protect neurons against oxidative injury. F1000Res 3: 28.

Ghate, P. S., Sidhar, H., Carlson, G. A. & Giri, R. K. (2014). Development of a novel cellular model of Alzheimer's disease utilizing neurosphere cultures derived from B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J embryonic mouse brain. Springerplus 3: 161.

Giffard, R. G. & Swanson, R. A. (2005). Ischemia-induced programmed cell death in astrocytes. Glia 50: 299-306.

Goetschy, J. F., Ulrich, G., Aunis, D. & Ciesielski-Treska, J. (1986). The organization and solubility properties of intermediate filaments and microtubules of cortical astrocytes in culture. J Neurocytol 15: 375-387.

Introduction 15

Grumbles, R. M., Almeida, V. W., Casella, G. T., Wood, P. M., Hemstapat, K. & Thomas, C. K. (2012). Motoneuron replacement for reinnervation of skeletal muscle in adult rats. J Neuropathol Exp Neurol 71: 921-930.

Guerout, N., Li, X. & Barnabe-Heider, F. (2014). Cell fate control in the developing central nervous system. Exp Cell Res 321: 77-83.

Hamprecht, B. & Löffler, F. (1985). Primary glial cultures as a model for studying hormone action. Methods Enzymol 109: 341-345.

Hansson, E. & Thorlin, T. (1999). Brain primary cultures and vibrodissociated cells as tools for the study of astroglial properties and functions. Dev Neurosci 21: 1-11.

Herculano-Houzel, S. (2014). The glia/neuron ratio: how it varies uniformly across brain structures and species and what that means for brain physiology and evolution. Glia 62:1377-1391.

Hertz, L., Schousboe, A., Boechler, N., Mukerji, S. & Fedoroff, S. (1978). Kinetic characteristics of the glutamate uptake into normal astrocytes in cultures. Neurochem Res 3: 1-14.

Hille, B. & Catterall, W. A. (2006). Electrical excitability and ion channels. In Basic Neurochemistry: molecular, cellular, and medical aspects 7 (Eds. Siegel, G. J., Albers, R. W., Brady, S. T. & Price, D. L.), Academic Press, Elsevier, pp. 95-109.

Holz, R. W. & Fisher, S. K. (2006). Synaptic transmission and cellular signaling: an overview. In Basic neurochemistry: molecular, cellular, and medical aspects 7 (Eds. Siegel, G. J., Albers, R. W., Brady, S. T. & Price, D. L.), Academic Press, Elsevier, pp. 167-183.

Howarth, C. (2014). The contribution of astrocytes to the regulation of cerebral blood flow. Front Neurosci 8: 103 (9 p.).

Jameson, N., Olson, J., Nguyen, H. & Holtzman, D. (1984). Respiration in primary cultured cerebellar granule neurons and cerebral cortical neurons. J Neurochem 42: 470-474.

Jarjour, A. A., Zhang, H., Bauer, N., Ffrench-Constant, C. & Williams, A. (2012). In vitro modeling of central nervous system myelination and remyelination. Glia 60: 1-12.

Jeon, H., Kim, J. H., Kim, J. H., Lee, W. H., Lee, M. S. & Suk, K. (2012). Plasminogen activator inhibitor type 1 regulates microglial motility and phagocytic activity. J Neuroinflammation 9: 149 (22 p.).

Jones, E. V., Cook, D. & Murai, K. K. (2012). A neuron-astrocyte co-culture system to investigate astrocyte-secreted factors in mouse neuronal development. Methods Mol Biol 814: 341-352.

Kang, K. S., Lee, S. I., Hong, J. M., Lee, J. W., Cho, H. Y., Son, J. H., Paek, S. H. & Cho, D. W. (2014). Hybrid scaffold composed of hydrogel/3D-framework and its application as a dopamine delivery system. J Control Release 175: 10-16.

Kettenmann, H., Hanisch, U. K., Noda, M. & Verkhratsky, A. (2011). Physiology of microglia. Physiol Rev 91: 461-553.

Kettenmann, H. & Verkhratsky, A. (2011). Neuroglia, der lebende Nervenkitt. Fortschr Neurol Psychiatr 79: 588-597.

Kimelberg, H. K. (2010). Functions of mature mammalian astrocytes: a current view. Neuroscientist 16: 79-106.

Kondratskaya, E., Shin, M. C. & Akaike, N. (2010). Neuronal glutamate transporters regulate synaptic transmission in single synapses on CA1 hippocampal neurons. Brain Res Bull 81: 53-60.

Lallouette, J., De Pitta, M., Ben-Jacob, E. & Berry, H. (2014). Sparse short-distance connections enhance calcium wave propagation in a 3D model of astrocyte networks. Front Comput Neurosci 8: 45 (18 p.).

16 Introduction

Lange, S. C., Bak, L. K., Waagepetersen, H. S., Schousboe, A. & Norenberg, M. D. (2012). Primary cultures of astrocytes: their value in understanding astrocytes in health and disease. Neurochem Res 37: 2569-2588.

Lau, C. L., Kovacevic, M., Tingleff, T. S., Forsythe, J. S., Cate, H. S., Merlo, D., Cederfur, C., Maclean, F. L., Parish, C. L., Horne, M. K., Nisbet, D. R. & Beart, P. M. (2014). 3D electrospun scaffolds promote a cytotrophic phenotype of cultured primary astrocytes. J Neurochem 130: 215-226.

Lee, H. U., Yamazaki, Y., Tanaka, K. F., Furuya, K., Sokabe, M., Hida, H., Takao, K., Miyakawa, T., Fujii, S. & Ikenaka, K. (2013). Increased astrocytic ATP release results in enhanced excitability of the hippocampus. Glia 61: 210-224.

Lee, Y., Morrison, B. M., Li, Y., Lengacher, S., Farah, M. H., Hoffman, P. N., Liu, Y., Tsingalia, A., Jin, L., Zhang, P. W., Pellerin, L., Magistretti, P. J. & Rothstein, J. D. (2012). Oligodendroglia metabolically support axons and contribute to neurodegeneration. Nature 487: 443-448.

Liu, T., Xue, C. C., Shi, Y. L., Bai, X. J., Li, Z. F. & Yi, C. L. (2014). Overexpression of mitofusin 2 inhibits reactive astrogliosis proliferation in vitro. Neurosci Lett 579: 24-29.

Marlatt, M. W., Bauer, J., Aronica, E., van Haastert, E. S., Hoozemans, J. J., Joels, M. & Lucassen, P. J. (2014). Proliferation in the Alzheimer hippocampus is due to microglia, not astroglia, and occurs at sites of amyloid deposition. Neural Plast 2014: 693851 (12 p.).

Matsui, T., Kida, H., Iha, T., Obara, T., Nomura, S., Fujimiya, T. & Suzuki, M. (2014). Effects of hypothermia on ex vivo microglial production of pro- and anti-inflammatory cytokines and nitric oxide in hypoxic-ischemic brain-injured mice. Folia Neuropathol 52: 151-158.

McCarthy, K. D. & de Vellis, J. (1980). Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol 85: 890-902.

Milligan, C. E., Cunningham, T. J. & Levitt, P. (1991). Differential immunochemical markers reveal the normal distribution of brain macrophages and microglia in the developing rat brain. J Comp Neurol 314: 125-135.

Monteiro, R. A., Henrique, R. M., Rocha, E., Marini-Abreu, M. M., Oliveira, M. H. & Silva, M. W. (1998). Age-related changes in the volume of somata and organelles of cerebellar granule cells. Neurobiol Aging 19: 325-332.

Morrison, B. M., Lee, Y. & Rothstein, J. D. (2013). Oligodendroglia: metabolic supporters of axons. Trends Cell Biol 23: 644-651.

Nayak, D., Roth, T. L. & McGavern, D. B. (2014). Microglia development and function. Annu Rev Immunol 32: 367-402.

Neumann, H. & Wekerle, H. (2013). Brain microglia: watchdogs with pedigree. Nat Neurosci 16: 253-255.

Norenberg, M. D. & Martinez-Hernandez, A. (1979). Fine structural localization of glutamine synthetase in astrocytes of rat brain. Brain Res 161: 303-310.

Norton, W. T. & Poduslo, S. E. (1973). Myelination in rat brain: changes in myelin composition during brain maturation. J Neurochem 21: 759-773.

Oberheim, N. A., Goldman, S. A. & Nedergaard, M. (2012). Heterogeneity of astrocytic form and function. Methods Mol Biol 814: 23-45.

Palmer, M. J., Taschenberger, H., Hull, C., Tremere, L. & von Gersdorff, H. (2003). Synaptic activation of presynaptic glutamate transporter currents in nerve terminals. J Neurosci 23: 4831-4841.

Pasca, S. P., Panagiotakos, G. & Dolmetsch, R. E. (2014). Generating human neurons in vitro and using them to understand neuropsychiatric disease. Annu Rev Neurosci 37: 479-501.

Introduction 17

Pedraza, C. E., Taylor, C., Pereira, A., Seng, M., Tham, C. S., Izrael, M. & Webb, M. (2014). Induction of oligodendrocyte differentiation and in vitro myelination by inhibition of rho-associated kinase. ASN Neuro 6 (17 p.).

Pelvig, D. P., Pakkenberg, H., Stark, A. K. & Pakkenberg, B. (2008). Neocortical glial cell numbers in human brains. Neurobiol Aging 29: 1754-1762.

Peters, D. G. & Connor, J. R. (2014). Introduction to cells comprising the nervous system. Adv Neurobiol 9: 33-45.

Pisanu, A., Lecca, D., Mulas, G., Wardas, J., Simbula, G., Spiga, S. & Carta, A. R. (2014). Dynamic changes in pro- and anti-inflammatory cytokines in microglia after PPAR-gamma agonist neuroprotective treatment in the MPTPp mouse model of progressive Parkinson's disease. Neurobiol Dis 71: 280-291.

Raine, C. S. (2006). Neurocellular anatomy. In Basic Neurochemistry: molecular, cellular, and medical aspects 7 (Eds. Siegel, G. J., Albers, R. W., Brady, S. T. & Price, D. L.), Academic Press, Elsevier, pp. 3-19.

Richter-Landsberg, C. & Heinrich, M. (1996). OLN-93: a new permanent oligodendroglia cell line derived from primary rat brain glial cultures. J Neurosci Res 45: 161-173.

Rinholm, J. E. & Bergersen, L. H. (2013). White matter lactate - does it matter? Neuroscience 276: 109-116.

Roberts, R. C., Roche, J. K. & McCullumsmith, R. E. (2014). Localization of excitatory amino acid transporters EAAT1 and EAAT2 in human postmortem cortex: a light and electron microscopic study. Neuroscience 277: 522-540.

Rodhe, J. (2013). Cell culturing of human and murine microglia cell lines. Methods Mol Biol 1041: 11-16.

Saura, J. (2007). Microglial cells in astroglial cultures: a cautionary note. J Neuroinflammation 4: 26 (11 p.).

Saura, J., Tusell, J. M. & Serratosa, J. (2003). High-yield isolation of murine microglia by mild trypsinization. Glia 44: 183-189.

Scalettar, B. A. (2006). How neurosecretory vesicles release their cargo. Neuroscientist 12: 164-176.

Sheng, Z. H. (2014). Mitochondrial trafficking and anchoring in neurons: new insight and implications. J Cell Biol 204: 1087-1098.

Silman, I. & Sussman, J. L. (2005). Acetylcholinesterase: 'classical' and 'non-classical' functions and pharmacology. Curr Opin Pharmacol 5: 293-302.

Sofroniew, M. V. & Vinters, H. V. (2010). Astrocytes: biology and pathology. Acta Neuropathol 119: 7-35.

Stansley, B., Post, J. & Hensley, K. (2012). A comparative review of cell culture systems for the study of microglial biology in Alzheimer's disease. J Neuroinflammation 9: 115 (8 p.).

Stevens, C. F. & Williams, J. H. (2000). "Kiss and run" exocytosis at hippocampal synapses. Proc Natl Acad Sci U S A 97: 12828-12833.

Sunol, C., Babot, Z., Fonfria, E., Galofre, M., Garcia, D., Herrera, N., Iraola, S. & Vendrell, I. (2008). Studies with neuronal cells: from basic studies of mechanisms of neurotoxicity to the prediction of chemical toxicity. Toxicol In Vitro 22: 1350-1355.

Szabo, M. & Gulya, K. (2013). Development of the microglial phenotype in culture. Neuroscience 241: 280-295.

Takacs, J. & Hamori, J. (1994). Developmental dynamics of Purkinje cells and dendritic spines in rat cerebellar cortex. J Neurosci Res 38: 515-530.

Tamashiro, T. T., Dalgard, C. L. & Byrnes, K. R. (2012). Primary microglia isolation from mixed glial cell cultures of neonatal rat brain tissue. J Vis Exp: e3814 (5 p.).

18 Introduction

Tanaka, T., Wakabayashi, T., Oizumi, H., Nishio, S., Sato, T., Harada, A., Fujii, D., Matsuo, Y., Hashimoto, T. & Iwatsubo, T. (2014). CLAC-P/collagen type XXV is required for the intramuscular innervation of motoneurons during neuromuscular development. J Neurosci 34: 1370-1379.

Tasaki, I. (1939). The electro-saltatory transmission of the nerve impulse and the effect of narcosis upon the nerve fiber. Am J Physiol 127: 211-227.

Thorburne, S. K. & Juurlink, B. H. (1996). Low glutathione and high iron govern the susceptibility of oligodendroglial precursors to oxidative stress. J Neurochem 67: 1014-1022.

Tulpule, K., Hohnholt, M. C., Hirrlinger, J. & Dringen, R. (2014). Primary cultures of astrocytes and neurons as model systems to study the metabolism and metabolite export from brain cells. In Neuromethods 90: Brain energy metabolism (Eds. Waagepetersen, H. & Hirrlinger, J.), Heidelberg, Springer, pp. 45-72.

Verkhratsky, A., Nedergaard, M. & Hertz, L. (2014). Why are astrocytes important? Neurochem Res, in press.

Verkhratsky, A. & Parpura, V. (2010). Recent advances in (patho)physiology of astroglia. Acta Pharmacol Sin 31: 1044-1054.

Virgintino, D., Monaghan, P., Robertson, D., Errede, M., Bertossi, M., Ambrosi, G. & Roncali, L. (1997). An immunohistochemical and morphometric study on astrocytes and microvasculature in the human cerebral cortex. Histochem J 29: 655-660.

Watkins, T. A., Emery, B., Mulinyawe, S. & Barres, B. A. (2008). Distinct stages of myelination regulated by γ-secretase and astrocytes in a rapidly myelinating CNS coculture system. Neuron 60: 555-569.

Weightman, A., Jenkins, S., Pickard, M., Chari, D. & Yang, Y. (2014). Alignment of multiple glial cell populations in 3D nanofiber scaffolds: toward the development of multicellular implantable scaffolds for repair of neural injury. Nanomedicine 10: 291-295.

Welser, J. V. & Milner, R. (2012). Use of astrocyte-microglial cocultures to examine the regulatory influence of astrocytes on microglial activation. Methods Mol Biol 814: 367-380.

Westerink, R. H. & Ewing, A. G. (2008). The PC12 cell as model for neurosecretion. Acta Physiol (Oxf) 192: 273-285.

Wilms, H., Hartmann, D. & Sievers, J. (1997). Ramification of microglia, monocytes and macrophages in vitro: influences of various epithelial and mesenchymal cells and their conditioned media. Cell Tissue Res 287: 447-458.

Wilson, J. X., Peters, C. E., Sitar, S. M., Daoust, P. & Gelb, A. W. (2000). Glutamate stimulates ascorbate transport by astrocytes. Brain Res 858: 61-66.

Wu, V. W. & Schwartz, J. P. (1998). Cell culture models for reactive gliosis: new perspectives. J Neurosci Res 51: 675-681.

Xie, H. R., Hu, L. S. & Li, G. Y. (2010). SH-SY5Y human neuroblastoma cell line: in vitro cell model of dopaminergic neurons in Parkinson's disease. Chin Med J (Engl) 123: 1086-1092.

Yoo, S. & Wrathall, J. R. (2007). Mixed primary culture and clonal analysis provide evidence that NG2 proteoglycan-expressing cells after spinal cord injury are glial progenitors. Developmental Neurobiology 67: 860-874.

Yu, A. C., Lee, Y. L. & Eng, L. F. (1993). Astrogliosis in culture: I. The model and the effect of antisense oligonucleotides on glial fibrillary acidic protein synthesis. J Neurosci Res 34: 295-303.

Zhuang, Z., Huang, J., Cepero, M. L. & Liebl, D. J. (2011). Eph signaling regulates gliotransmitter release. Commun Integr Biol 4: 223-226.

Introduction 19

1.4. PPublication 1

Uptake and metabolism of iron oxide nanoparticles in brain cells

Charlotte Petters, Ellen Irrsack, Michael Koch, Ralf Dringen

Neurochem Res (2014) 39:1648-1660

Contributions of Charlotte Petters

1st draft of chapters “Synthesis and properties of iron oxide nanoparticles” and “Uptake, metabolism and toxicity of iron oxide nanoparticles in cultured brain cells”

Preparation of figures 1-5 and table 1 Experimental work for figure 5 Improvement of the manuscript

1.4 Publication 1

Introduction 21

The pdf-document of this publication is not displayed due to copyright reasons. The publication can be assessed at: http://link.springer.com/article/10.1007%2Fs11064-014-1380-5; DOI: 10.1007/s11064-014-1380-5.

22 Introduction

Introduction 23

1.5. PPublication 2

Handling of iron oxide and silver nanoparticles by astrocytes

Michaela C. Hohnholt, Mark Geppert, Eva M. Luther, Charlotte Petters,

Felix Bulcke, Ralf Dringen

Neurochem Res (2013) 38:227-239

Contributions of Charlotte Petters

First draft of “Conclusions and Perspectives” Table 2: Information on particle characteristics, culture, and research aspects

Improvement of the maunscript

1.5 Publication 2

Introduction 25

The pdf-document of this publication is not displayed due to copyright reasons. The

publication can be assessed at: http://link.springer.com/article/10.1007%2Fs11064-012-

0930-y; DOI: 10.1007/s11064-012-0930-y.

Introduction 27

1.6. AAim of the thesis

The aim of this thesis is to investigate IONP uptake and localization of internalized

IONPs in brain cell cultures by using fluorescent IONPs. Firstly, fluorescently labelled

IONPs will be prepared and characterized for their physicochemical properties including

parameters such as size, stability and fluorescence. Furthermore, these fluorescent IONPs

will be compared with their non-fluorescent counterpart for their physicochemical

properties but also for their uptake into brain cells. Finally the characterized IONPs will

be used to compare the accumulation, localization and potential adverse effects of IONPs

on different types of culture brain cells.

2. RResults

2.1. Publication 3 ........................................................................................................ 31

Petters C, Dringen R (2014) Comparison of primary and secondary rat astrocyte cultures regarding glucose and glutathione metabolism and the accumulation of iron oxide nanoparticles. Neurochem Res 39:46-58

2.2. Publication 4 ........................................................................................................ 35

Petters C, Bulcke F, Thiel K, Bickmeyer U, Dringen R (2014) Uptake of fluorescent iron oxide nanoparticles by oligodendroglial OLN-93 cells. Neurochem Res 39:372-383

2.3. Publication 5 ........................................................................................................ 39

Luther EM, Petters C, Bulcke F, Kaltz A, Thiel K, Bickmeyer U, Dringen R (2013) Endocytotic uptake of iron oxide nanoparticles by cultured brain microglial cells. Acta Biomater 9:8454-8465

2.4. Publication 6 ........................................................................................................ 43

Petters C, Dringen R (2015) Accumulation of iron oxide nanoparticles by cultured primary neurons. Neurochem Int 81:1-9

2.5. Publication 7 (manuscript) ................................................................................... 47

Petters C, Dringen R Lysosomal iron liberation is responsible for the high vulnerability of brain microglial cells to iron oxide nanoparticles: comparison with neurons and astrocytes. Submitted

Results 31

2.1. PPublication 3

Comparison of primary and secondary rat astrocyte

cultures regarding glucose and glutathione

metabolism and the accumulation of iron oxide

nanoparticles

Charlotte Petters, Ralf Dringen

Neurochem Res (2014) 39:46–58

Contributions of Charlotte Petters

Design of the study Experimental work

Preparation of the first draft of the manuscript

2.1 Publication 3

Results 33

The pdf-document of this publication is not displayed due to copyright reasons. The publication can be assessed at: http://link.springer.com/article/10.1007%2Fs11064-013-1189-7; DOI: 10.1007/s11064-013-1189-7.

Results 35

2.2. PPublication 4

Uptake of fluorescent iron oxide nanoparticles by oligodendroglial OLN-93 cells

Charlotte Petters, Felix Bulcke, Karsten Thiel, Ulf Bickmeyer, Ralf Dringen

Neurochem Res (2014) 39:372-383

Contributions of Charlotte Petters

Design of the study Experimental work for figures 1g,h, 2-6, table 1 and 2

First draft of the manuscript

2.2 Publication 4

Results 37

The pdf-document of this publication is not displayed due to copyright reasons. The

publication can be assessed at: http://link.springer.com/article/10.1007%2Fs11064-013-

1234-6; DOI: 10.1007/s11064-013-1234-6.

38 Results

Results 39

2.3. PPublication 5

Endocytotic uptake of iron oxide nanoparticles by cultured brain microglial cells

Eva M. Luther, Charlotte Petters, Felix Bulcke, Achim Kaltz, Karsten Thiel, Ulf Bickmeyer, Ralf Dringen

Acta Biomater (2013) 9:8454-8465

Contributions of Charlotte Petters

Experimental work for figure 1 and table 1 First draft of text concerning synthesis and characterization of IONPs

Preparation of figures S1 and S2

2.3 Publication 5

Results 41

The pdf-document of this publication is not displayed due to copyright reasons. The

publication can be assessed at:

http://www.sciencedirect.com/science/article/pii/S1742706113002717; DOI:

10.1016/j.actbio.2013.05.022.

42 Results

Results 43

2.4. PPublication 6

Accumulation of iron oxide nanoparticles by cultured primary neurons

Charlotte Petters, Ralf Dringen

Neurochem Int 81:1-9

Contributions of Charlotte Petters

Design of the study Experimental work

First draft of the manuscript

2.4 Publication 6

Results 45

The pdf-document of this publication is not displayed due to copyright reasons. The

publication can be assessed at:

http://www.sciencedirect.com/science/article/pii/S0197018614002526; DOI:

10.1016/j.neuint.2014.12.005.

46 Results

Results 47

2.5. PPublication 7 (manuscript)

Lysosomal iron liberation is responsible for the high vulnerability of brain microglial cells to iron oxide

nanoparticles: comparison with neurons and astrocytes

Charlotte Petters, Ralf Dringen

submitted

Contributions of Charlotte Petters

Design of the study Experimental work

First draft of the manuscript

2.5 Publication 7 (manuscript)

Results 49

LLysosomal iron liberation is responsible for the high vulnerability of brain microglial cells to iron oxide nanoparticles: comparison with neurons and astrocytes

Charlotte Petters1, 2, Ralf Dringen1, 2

1Center for Biomedical Interactions Bremen, University of Bremen, PO. Box 330440, D-

28334 Bremen, Germany.

2Center for Environmental Research and Sustainable Technology, Leobener Strasse, D-

28359 Bremen, Germany.

Address correspondence to: Dr. Ralf Dringen Center for Biomolecular Interactions Bremen University of Bremen PO. Box 330440 D-28334 Bremen Germany Tel: +49-421-218- 63230 Fax: +49-421-218-63244 Email: ralf.dringen@uni-bremen.de homepage: http://www.fb2.uni-bremen.de/en/dringen

Keywords: Nanotoxicology, Biocompatibility, Biochemistry

50 Results

AAbstract

Iron oxide nanoparticles (IONPs) are used for various biomedical and neurobiological

applications. Thus, detailed knowledge on the accumulation and toxic potential of IONPs

for the different types of brain cells is highly warranted. Literature data suggest that

microglial cells are more vulnerable towards IONPs exposure than other types of brain

cells. To investigate the mechanisms involved in IONP-induced microglial toxicity we

applied fluorescent dimercaptosuccinate-coated IONPs to primary cultures of microglial

cells. Exposure to IONPs for 6 h caused a strong concentration-dependent increase in the

microglial iron content which was accompanied by a substantial generation of reactive

oxygen species (ROS) and by cell toxicity. In contrast, hardly any ROS staining and no

loss in cell viability were observed for cultured primary astrocytes and neurons although

these cultures accumulated similar specific amounts of IONPs than microglia. Co-

localization studies with lysotracker revealed that in microglial cells, but not in astrocytes

and neurons, most IONP fluorescence was localized in lysosomes. ROS formation and

toxicity in IONP-treated microglial cultures were prevented by neutralizing lysosomal pH

by application of NH4Cl or Bafilomycin A1 and by application of the iron chelator 2,2’-

bipyridyl. These data demonstrate that rapid iron liberation from IONPs at acidic pH

and iron-catalyzed ROS generation are involved in the IONP-induced toxicity of

microglia and suggest that the relative resistance of astrocytes and neurons against acute

IONP toxicity is a consequence of a slow mobilization of iron from IONPs in the

lysosomal degradation pathway.

Results 51

IIntroduction

Iron oxide nanoparticles (IONPs) have gained substantial attention for biomedical

applications and are already used as contrast agent in magnetic resonance imaging (MRI),

for cancer treatment by hyperthermia or for targeted drug delivery (Li et al., 2013;

Mahmoudi et al., 2011; Maier-Hauff et al., 2011). Nanoparticles can enter the brain via

the intact or damaged blood-brain barrier, via the olfactory nerve or by direct injection

into brain in the case of hyperthermia treatment (Petters et al., 2014b). By such

applications all types of brain cells are likely to encounter IONPs in vivo.

In addition to neurons which are responsible for signal transduction, transmit information

along their axon and release neurotransmitters to other neurons (Peters and Connor,

2014; Scalettar, 2006), the brain contains various types of glial cells in substantial numbers

(Peters and Connor, 2014). Microglial cells are the immune competent cells of the brain

and clear the brain from cell debris and intruders (Nayak et al., 2014; Eyo and Dailey,

2013). In case of incident, microglial cells become activated, transform from their resting

ramified into their amoeboid form and migrate to the site of interference (Kettenmann et

al., 2011; Nayak et al., 2014). Astrocytes cover with their processes most of the brain

capillaries which form the blood-brain barrier (De Bock et al., 2014), supply other brain

cell types with nutrients (Bouzier-Sore and Pellerin, 2013; Stobart and Anderson, 2013),

are responsible for the homeostasis of ions, water and metals (Verkhratsky et al., 2014;

Dringen et al., 2013; Hohnholt and Dringen, 2013) and have important detoxifying

functions in brain (Fernandez-Fernandez et al., 2012; Dringen et al., 2015).

Several studies have investigated the uptake and biocompatibility of IONPs in culture

models for the different types of brain cell, including primary cultures of astrocytes,

neurons and microglial cells (for overview see: (Petters et al., 2014b)). However, data on a

quantitative comparison of the uptake and cytotoxicity of IONPs in different types of

brain cells are scarce. In brain cell culture systems, microglial cells have been reported to

take up fluorescent nanospheres and IONPs more efficiently than astrocytes and neurons

(Jenkins et al., 2013; Pinkernelle et al., 2012; Fernandes and Chari, 2014). In addition, in

co-cultures IONP uptake into astrocytes and oligodendrocyte precursor cells was lowered

in the presence of microglial cells (Pickard and Chari, 2010). In vivo, mainly macrophage-

like cells were found IONP-positive after injection of IONPs into human glioma (van

Landeghem et al., 2009), supporting the hypothesis that microglial cells have also in vivo a

high potential to accumulate IONPs. Macropinocytosis and clathrin-mediated endocytosis

52 Results

have been identified as molecular mechanisms involved in the IONP uptake by microglial

cells which direct the accumulated IONPs into the lysosomal degradation pathway

(Luther et al., 2013).

The strong accumulation of IONPs by microglia has been connected with a high

vulnerability of these cells towards IONPs (Jenkins et al., 2013; Luther et al., 2013) while

astrocytes (Geppert et al., 2012; Geppert et al., 2011; Jenkins et al., 2013) and neurons

(Sun et al., 2013; Petters and Dringen, 2015) appear to be relative resistant against acute

IONP toxicity. However, the mechanisms involved in the toxicity of IONPs for microglial

cells and the reason for the apparent resistance of astrocytes and neurons against IONPs

are currently unknown. To address such questions, we have exposed primary cultures of

microglia, neurons and astrocytes under identical experimental conditions to fluorescent

IONPs and directly compared the potential of the different neural cells for IONP

accumulation as well as the potential of IONPs to compromise cell viability. Here, we

report that cultured microglial cells accumulated low concentrations of fluorescent IONP

more efficiently than astrocytes and neurons while high concentrations of IONPs caused

severe ROS production and toxicity in microglial cells but not in astrocytes and neurons.

IONP-induced damage of microglial cells was prevented by neutralization of the acidic

lysosomal pH or by chelation of iron ions, suggesting that in microglial cells the rapid

lysosomal mobilization of iron from internalized IONPs causes accelerated ROS

formation and oxidative cell damage.

Results 53

MMaterial and methods

Materials

Dulbecco’s modified Eagle’s medium (DMEM) and minimal essential media (MEM)

were from Gibco (Karlsruhe, Germany). Fetal calf serum (FCS) and

penicillin/streptomycin solution were purchased from Biochrom (Berlin, Germany).

Bovine serum albumin and NADH were from Applichem (Darmstadt, Germany) and

Bafilomycin A1 was purchased from Invivogen (Toulouse, France). BODIPY FL C1-IA

(BP) [N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-

yl)methyl)iodoacetamide] and lysotracker red DND-99 were from Life Technologies

(Darmstadt, Germany). Other chemicals of the highest purity available were obtained

from Merck (Darmstadt, Germany), Fluka (Buchs, Switzerland) or Sigma-Aldrich

(Steinheim, Germany). 96-well microtiter plates and 6-well cell culture plates were

purchased from Sarstedt (Nümbrecht, Germany) and Nunc (Wiesbaden, Germany),

respectively.

Synthesis and characterization of fluorescent IONPs

Fluorescent dimercaptosuccinate (DMSA)-coated IONPs were synthesized and

characterized as previously reported (Petters et al., 2014a). Briefly, maghemite IONPs

were generated by an alkaline co-precipitation method (Geppert et al., 2009) and the

IONPs were coated with BP-functionalized DMSA (Petters et al., 2014a). Single

fluorescent IONPs were spherical and had a size of 4-20 nm as seen by transmission

electron microscopy (Petters et al., 2014a). The fluorescent BP-DMSA-IONPs emit light

at 510 nm after excitation at 490 nm (Petters et al., 2014a).

The hydrodynamic diameter and the ζ-potential of IONPs in suspension were

determined by dynamic and electrophoretic light scattering in a Beckman Coulter

(Krefeld, Germany) DelsaTM Nano Particle analyzer at 25°C at scattering angle of 165° and

15°, respectively. Concentrations of IONPs refer to the total concentration of iron in

dispersion and not to the concentration of nanoparticles.

Cell culture

Microglial cultures were generated by tryptic removal of the astrocyte layer of astrocyte-

rich primary cultures in 6-well plates one day prior to experiments as described previously

(Luther et al., 2013). The remaining attached microglia were washed once with culture

54 Results

medium (90% DMEM, 10% fetal calf serum, 25 mM glucose, 1 mM pyruvate, 20

units/mL penicillin G and 20 μg/mL streptomycin sulfate) and incubated in 2 mL glia-

conditioned medium (GCM) before incubations with IONPs were started.

Immunocytochemical characterization of the microglial cultures revealed that more than

98% of the cells in these cultures are positive for the microglial marker protein CD11b

(Luther et al., 2013).

Astrocyte-rich primary cultures were prepared from newborn Wistar rats as described

recently in detail (Tulpule et al., 2014). Cells were seeded at a density of 375,000

cells/well in 6-well plates and used at day five in culture for experiments. Astrocyte-rich

cultures are strongly enriched in cells positive for the astrocyte marker protein glial

fibrillary acidic protein, contain some microglial and oligodendroglial cells but no neurons

(Petters and Dringen, 2014; Tulpule et al., 2014).

Cerebellar granule neuron cultures were prepared from brains of 7 d-old Wistar rats as

described recently in detail (Tulpule et al., 2014). The cells were seeded in neuron

culture medium (90% MEM, 10% heat-inactivated FCS, 25 mM KCl, 30 mM glucose and

2 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin) in a density of

1,875,000 cells/well in 6-well plates. Neurons were used for experiments after seven days

in culture. Immunocytochemical characterization of the neuron cultures revealed that

99% of the cells expressed the neuronal marker protein microtubule-associated protein-2

(Tulpule et al., 2014).

Experimental incubations

GCM is required to avoid toxicity of microglial cells in cultures (Saura et al., 2003). In

order to establish comparable incubation conditions for primary microglial cells, neurons

and astrocytes, all primary cultures were incubated in GCM. GCM was prepared as

previously described (Luther et al., 2013) by incubating astrocyte-rich primary cultures in

175 cm2 flasks for 1 d with 50 mL culture medium. The medium was harvested, filtered

through a 0.2 μm sterile filter and stored at 4°C for not more than three weeks (Luther et

al., 2013). In case of experiments on cerebellar granule neurons, the GCM had to be

supplemented with 25 mM KCl to prevent neuronal apoptosis (Contestabile, 2002).

To investigate acute consequences of IONPs on cultured primary neural cells, the

cultures were incubated at 37°C in a humidified atmosphere with 10% CO2 in 1 mL GCM

Results 55

without or with IONPs and/or other compounds as indicated in the legends of the figures

and tables.

After incubation, cells were washed once with 1 mL phosphate-buffered saline (PBS, 10

mM potassium phosphate buffer, pH 7.4, containing 150 mM NaCl) and lysed in 1 mL

1% (w/v) Triton X-100 in PBS to measure cellular lactate dehydrogenase (LDH) activity

or in 700 μL 50 mM NaOH to determine cellular protein and iron contents.

For cellular localization of fluorescent IONPs, cultures were incuba