U
Uptake 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
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
T
ABLE OF
C
ONTENT
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
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
II. A
CKNOWLEDGEMENTS
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.
III. S
TRUCTURE 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.
IIII. S
UMMARY
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
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.
IIV. Z
USAMMENFASSUNG
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
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.
V
V. A
BBREVIATIONS 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)
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
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 ... 19Petters 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
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).
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).
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).
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).
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).
1.2. C
Culture 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
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).
F
Figure 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.
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
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).
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.
1.3. R
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1.4. P
Publication 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
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.
1.5. P
Publication 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
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.
1.6. A
Aim 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. R
Results
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
2.1. P
Publication 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
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.
2.2. P
Publication 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
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.
2.3. P
Publication 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
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.
2.4. P
Publication 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
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.
2.5. P
Publication 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)
L
Lysosomal 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 1
Center for Biomedical Interactions Bremen, University of Bremen, PO. Box 330440, D-28334 Bremen, Germany.
2
Center 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
A
Abstract
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.
