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«Dissertation Zur Erlangung des Grades Doktor der Naturwissenschaften Am Fachbereich Biologie Der Johannes Gutenberg-Universität Mainz Vorgelegt von ...»

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Local Self-Renewing of Microglia after

Genetic Ablation is Dependent on

Interleukin-1 Signaling

Dissertation

Zur Erlangung des Grades

Doktor der Naturwissenschaften

Am Fachbereich Biologie

Der Johannes Gutenberg-Universität Mainz

Vorgelegt von

Julia Bruttger

geb. am 14. Juni 1984 in Bad Kreuznach, Deutschland

Mainz, 2015

Tag der mündlichen Prüfung: __25.01.2016_________________________

Für Sven

Table of Contents

Table of Contents

Table of Contents

Abbreviations

1 Introduction

1.1 Microglia – The Link between Nervous System and Immune System

1.1.1 Glia Cells and the Central Nervous System

1.1.2 Microglia and their Role in the Immune System

1.2 Microglia - Factors on which they Depend on

1.3 Development of Microglia

1.4 Microglia Depletion as Research Model

1.4.1 Full Deletion of Single Genes in Mice

1.4.2 Conditional Genetic Deletion Models

1.4.3 Pharmacological Treatment for Microglia Ablation

1.5 Aim of the Study

1 2 Materials and Methods

2.1 Chemicals and Biological Material

2.2 Molecular Biology

2.2.1 Isolation of Genomic DNA

2.2.2 Polymerase Chain Reaction (PCR)

2.2.3 Agarose Gel Electrophoresis

2.2.4 RNA Isolation

.

2.2.5 Quantitative Real Time PCR

2.2.6 Next Generation Sequencing

2.3 Cell biology

2.3.1 Preparation of Single Cell Suspension from CNS Tissue

2.3.2 Cell Counting

2.3.3 Flow Cytometry

.

2.3.4 Magnetic Cell Sorting and FACS Sorting

2.4 Histological Analysis and Immunohistochemistry

2.4.3 Quantification of Histology

2.5 Mouse Experiments

2.5.1 Mice

2.5.2 Tamoxifen Treatment

2.5.3 Diphtheria Toxin Treatment

2.5.4 Antibody Treatment

2.5.5 BrdU Feeding

2.5.6 Intracerebroventricular Treatment with IL-1RA

2.5.7 Bone Marrow Transplantation

2.5.8 EAE Induction

2.5.9 Statistics

2.5.9 NGS Data Anlysis

3 Results

3.1 Targeting Microglia with the CX3CR1CreER System

3.1.1 Microglia are Efficiently Depleted using the CX3CR1CreER System

3.1.2 Microglia Ablation Leads to Fast Repopulation Within Four Days

3.1.3 Microglia Depletion Leads to Self Activation, Astrogliosis and Cytokine Storm...... 32

3.2 BM-derived Macrophages Contribute to Microglia Pool in BM Chimeric Mice..... 6 3 3.2.1 Cells with BM Origin Repopulate the CNS

3.2.2 Cells with BM Origin Repopulate the CNS and Stay for Long

I Table of Contents 3.2.3 HSCs Can also be Excluded as a Peripheral Source of Microglia

3.3 In Absence of Irradiation Microglia Renew Exclusively from Internal Pools........ 1. 4 3.3.1 Clusters of Proliferating Microglia Repopulate the CNS

3.3.2 Newly Repopulated CNS-derived Microglia Display Unaltered Gene Expression Profiles, While BM Macrophages are Distinct

3.4 Factors that Influence Microglia Repopulation

3.4.1 Nestin is Expressed in Repopulating Microglia

3.4.2 Csf-1 and Its Receptors are Important Players which Differentiate Microglia from BM Macrophages

3.4.3 Microglia Repopulation is Dependent on Interleukin-1 Signaling

3.4.4. IL-1RA Blockade Effects Microglia Proliferation early after I.C.V. Treatment......... 57 3.4.5 Impact of Depletion and IL-1RA Treatment on Other Brain Resident Cells.............. 60 3.4.6 Conditional K.O. of IL-1R1 on Microglia Effects Microglia Maintenance

3.4.7 Disease Onset of EAE is Delayed in IL-1RMG mice

4 Discussion

4.1 Microglia specific targeting

6

4.2 Cx3Cr1CreER:iDTR System: A Model for Genetic Depletion of Microglia

4.3 The CD11b-HSVTK System

4.4 Pharmacological Depletion of Microglia

4.5 Microglia Repopulation

7

4.6 Microglia Progenitors vs. Self-maintenance

4.7 Distinct RNA-Seq Profiles of Different Microglia Populations

4.8 Impact of Interleukin-1 on Microglia

5 Summary

6 Zusammenfassung

7 References

8 Erklärung

9 Publications

–  –  –

IL-1RA Interleukin-1 receptor antagonist IL-12β Interleukin 12-beta IL-34 Interleukin 34 i.c.v. intra-cerebro-ventricular iDTR inducible diphtheria toxin receptor i.p. intra-peritoneal i.v. intra-veneously Irf8 interferon regulatory factor 8 Mac-1 macrophage-1 antigen or CD11b MFI mean Fluorescence Intensity

–  –  –

Myb myeloblastosis oncogene Nos2 inducible nitric oxide synthase 2 PCA principle component analysis PNS peripheral nervous system

–  –  –

1 Introduction

1.1 Microglia – the Link between Nervous System and Immune System 1.1.1 Glia Cells and the Central Nervous System The human nervous system consists of the central nervous system (CNS) and the peripheral nervous system (PNS). The CNS is composed of the brain and spinal cord, whereas the PNS encompasses the nervous tissues outside of the CNS. The human brain is highly complex, consisting of approximately 1 x 1012 neurons, where one neuron can form as many as 1000 synaptic connections. This constitutes anything from simple motor controls to more sophisticated emotional and cognitive behavior. Supporting these functions, the spinal cord integrates responses to different types of stimuli that are conveyed to and from the brain. The PNS is essential for monitoring the internal environment, by relaying information to the CNS.





The term “Glia” was introduced by Rudolf Virchow in 1859 and implies “nerve glue”. It was believed that glial cells only serve as an agglutinative unit giving structural support. In fact, glia cells provide a much more crucial role in the CNS than just structural support. They outnumber the neurons by tenfold, maintaining the functionality of this intricate neuronal network in many different ways (Baumann and Pham-Dinh, 2001). Glia cells are divided into two groups: the macroglia consisiting of astrocytes, oligodendrocytes, and oligodendrocyte precursor cells, and the microglia (Fig. 1). Astrocytes supply neurons with nutrients from the blood circulation. They regulate the extracellular ion concentration, are capable of recycling neurotramsmitters (Miller, 2002) and they support synaptogenesis and the blood-brain barrier (Ballabh et al., 2004). Oligodendrocytes form the myelin sheath around the axons. In the CNS, one oligodendrocyte is able to myelinate up to 50 axonal segments (Bjartmar et al., 1994) whereas one Schwann cell in the PNS typically myelinates only a single axon (Salzer, 2003). During myelination the oligodendrocyte ensheaths the axon several times, resulting in electrical

1 Introduction

insulation, thereby helping to accelerate the neuronal signal conduction.

Nodes of Ranvier arise in between the single myelinated segments allowing the signals to jump from node to node (internodes) resulting in what is known as saltatory conduction. In contrast to their progeny, oligodendrocyte precursor cells are much less understood. Besides generating mature oligodendrocytes, they are the only known glial cells that can form functional synapses with neurons. The reason why they form these synapses is still elusive, but is believed to help maintaining neuronal network functionality (Trotter et al., 2010). Microglia are the smallest cells among the glial cells.

They are CNS-resident myeloid cells that adopt various different functions.

Robertson (1900) and Nissl (1898) were the first to report these cells, describing them as “rod cells” (“Stäbchenzellen” in German) because of their rod-like shape, and noting their accumulation near inflammatory lesions in the CNS. Robertson was also the first to describe the phagocytic function of microglia: using Scharlach Red and hematoxylin staining, he was able to visualize fatty degeneration products that most likely resulted from destroyed myelin and neurons inside microglia. In 1919, Pio del Rio-Hortega, a Spanish scientist considered by many to be “the father of microglia”, provided an exceptional visualization of nervous tissue by inventing the silver carbonate method. He was also the first to name these cells “microglia” or the “third element”. Del Rio-Hortega was not only capable of distinguishing microglia from oligodendrocytes and astrocytes, he also discovered their potential to change morphology from ramified to amoeboid under pathological conditions (Del Rio-Hortega, 1932; Kettenmann et al., 2011; Samokhvalov et al., 2007).

–  –  –

Figure 1 Glia- neuron interactions (Allen and Barres, 2009) Different types of glia cells interacting with each other are shown. Neurons form synapses, which are controlled by astrocytes. On the other hand astrocytes are in contact with blood vessels. Oligodendrocytes form the myelin sheath by wrapping around the axons. Microglia survey the brain for damage or infection.

1.1.2 Microglia and their Role in the Immune System Microglia, as tissue resident macrophages, have crucial roles in tissue homeostasis and immunity. They are the only immune cells present in the healthy CNS parenchyma and are thus the first responders to any environmental change. Under conditions of CNS tissue damage, such as bacterial or viral infections, microglia play a critical role in clearing debris, leading to restoration of the CNS homeostasis (Koizumi et al., 2007; Sierra et al., 2010). Microglia function not only as innate immune cells in pathological conditions, but they also maintain tissue integrity in non-inflammatory

3 Introduction

conditions, such as secreting growth factors and anti-inflammatory molecules to helo in regeneration of damaged tissue (Davalos et al., 2005b; Kettenmann et al., 2011; Nimmerjahn et al., 2005; Prinz and Priller, 2014). It is clear that microglia are anything but “resting” in steady state: for immune surveillance they constantly extend and retract their processes to monitor their proximal environment for tissue damage or foreign invaders (Davalos et al., 2005a;

Nimmerjahn et al., 2005). In addition, emerging data reveal new and fundamental roles for microglia in the control of neuronal proliferation, differentiation and maintaining neuronal network integrity in the form of supporting synaptogenesis/ synaptic pruning (Paolicelli et al., 2011). This way, microglia are involved in building up the neuronal network, especially during brain development, and are capable to engulf and clear malformed synapses.

1.2 Microglia - Factors on which they Depend on

In previous studies the role of microglia in development, maintenance and inflammation, among other aspects, has been analyzed. Various molecules, such as transcription factors, chemokines and their receptors and growth factors are involved in shaping microglial phenotypes (Fig. 2). The following chapter will summarize the published knowledge about microglia in health and disease accompanied by known key factors controlling microglial genesis and homeostasis.

Endogenous transcription factors play critical roles in microglia development.

During embryogenesis, maturation and differentiation states of microglia are transcriptionally controlled by Runt-related transcription factor I (Runx1), interferon regulatory factor 8 (Irf8) and ETS (E-twenty six) family transcription factor PU.1 (Kierdorf and Prinz, 2013). Runx1 has a crucial role in definitive hematopoiesis since it is expressed early on by hematopoietic stem cells (HSCs) in the yolk sac (Ginhoux et al., 2010). Runx1 deficient mice completely lack HSCs and the deletion is embryonically lethal at the developmental stage E12.5. Runx1 is therefore considered as the major transcription factor driving the lineage of hematopoiesis, including microglia

4 Introduction

development. However, the function of Runx1 in microglia homeostasis in the adult CNS is still elusive. Postnatal, it is responsible for regulating microglia proliferation and activation status (Zusso et al., 2012). Runx1 also interacts with the other myeloid specific factors PU.1 and IRF8. PU.1 is essential for adult myelopoiesis (Hoeffel et al., 2015) and is expressed on early microglial progenitors, but is dispensable for proper HSC development (Schulz et al., 2012b). PU.1 deficiency leads to a lack of B cells along with complete suppression of myeloid cells including tissue macrophages like Kupffer cells or microglia. Thus, PU.1-deficient mice die shortly after birth. Furthermore, a deletion of PU.1 leads to down-regulation of Irf8 (Kierdorf et al., 2013c).

Additionally, PU.1 and Irf8 can act simultaneously as hetero-dimerization partners or Irf8 can function as downstream target of PU.1. However, there is no direct evidence if PU.1 is also involved in normal homeostasis of adult microglia.

Figure 2 Markers and morphology of embryonic and adult microglia (Ginhoux and Prinz, 2015) Embryonic microglia (left) show an activated and amoeboid phenotype. During adulthood microglia (right) exhibit small and delineated processes that actively survey the brain parenchyma. They also interact with local neighboring neurons. All transcription factors (black), receptors (red) and precursor stages are illustrated for each developmental stage.

5 Introduction

The chemokine receptor Colony stimulating factor 1 receptor (CSF-1R) is a tyrosine kinase transmembrane receptor mainly expressed by myeloid cells including microglia. CSF1R-/- mice lack microglia and epidermal Langerhans cells (Ginhoux and Merad, 2010; Ginhoux et al., 2006), but also exhibit broad myelo-suppression, impacting peripheral cells including macrophages, HSCs, osteoclasts and mast cells (Cornelis et al., 2005). A ligand for CSF-1R is the colony stimulating factor 1 (CSF-1), which is a critical factor for the proliferation, differentiation and survival of myeloid cells (Greter et al., 2012).



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