Characterisation of Angiogenin Uptake and Signalling in Astrocytes Alexandra Skorupa Royal College of Surgeons in Ireland

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1-1-2011 Characterisation of angiogenin uptake and signalling in astrocytes Alexandra Skorupa Royal College of Surgeons in Ireland

Citation Skorupa, A. Characterisation of angiogenin uptake and signalling in astrocytes. [PhD Thesis]. Dublin: Royal College of Surgeons in Ireland; 2011.

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This thesis is available at e-publications@RCSI: http://epubs.rcsi.ie/phdtheses/47 Characterisation of Angiogenin Uptake and Signalling in Astrocytes

Alexandra Skorupa

A Thesis Submitted to the Royal College of Surgeons in Ireland in Fulfilment of the Requirements for the Degree of Doctor of Philosophy

November 201 1

Department of Physiology and Medical Physics

Project Supervisors: Prof. Dr. Jochen Prehn and

Dr. Philippe Marin

I declare that this thesis, which I submit to RCSl for examination in consideration of the award of a higher degree, Doctor of Philosophy, is my own personal effort. Where any of the content presented is the result of input or data from related collaborative research program this is duly acknowledged in the text such that it is possible to ascertain how much of the work is my own. I have not already obtained a degree in RCSl or elsewhere on the basis of this work. Furthermore, I took reasonable care to ensure that the work is original, and, to the best of my knowledge, does not breach copyright law, and has not been taken from other sources except where such work has been cited and acknowledged within the text.

RCSl Student Number

Date 0

To my parents for their unending supporf

Content

Candidate Thesis Declaration

IP Declaration

Dedication

Table of Contents

Acknowledgements

Summa y in English

R&sume en Fran~ais

Publications and Presentations

Ab brevjations

1. Introduction

1.1 Amyotrophic Lateral Sclerosis 1.1. I Aetiology and Symptoms 1.I -2 Familial ALS Mutations .4 .I-3 SOD1 Mutant Toxicity I.I .4 Neurodegeneration in ALS 1.I .5 Cellular Players in ALS Pathology 1.I .6 Astrocytes Secrete Toxic and Protective Factors II. Astrocytes Specifically Interact with Motoneurons

1.2Angiogenin 1.2.1 Discovery 1.2.2 Molecular Properties 1.2.3 Angiogenin Internalisation 1.2.4 Signalling 1.2.5 Ribonucleolytic Activity 1.2.6 Angiogenin in Angiogenesis I.2.7 Angiogenin Mutations in ALS 1.2.8 Angiogenin in ALS Pathomechanism 1.2.9 Angiogenin Effects on Motoneurons

1.3Aims of the Study

2. Materials and Methods 32

2.1 Materials 2.1 .I Reagents 2.1.2 Equipment

2.2 Methods 36 2.2.1 Cell Culture 36 2.2.1 .I Preparation of Primary Motoneuron Cultures 36 2.2.1 -2 Preparation of Primary Astrocyte Cultures 3 8 2.2.1.3 NSC34 Cells 3 9 2.2.2 Angiogenin Uptake Studies 40 2.2.2.1 Organelle Markers to Monitor lntracellular Trafficking 2.2.2.2 Inhibition of Endocytosis 2.2.3 lmmunocytochemistry 2.2.3.1 lmmunostaining 2.2.3.2 Fluorescence Microscopy to Visualise lmmunocytochemistry 2.2.4 Electron Microscopy 2.2.4.1 Cell Fixation and Embedding 2.2.4.2 Sectioning and Staining 2.2.5 Plasmids and Transfection 2.2.5.1 Cell Transfection 47 2.2.5.2 Plasmid Amplification 48 2.2.5.3 Protein Overexpression 49 2.2.5.4 Protein Knock Down 5 0 2.2.6 Western Blotting 5 1 2.2.6. I Sample Preparation 52 2.2.6.2 Gel Preparation 53 2.2.6.3 Gel Electrophoresis 54 2.2.6.4 Semi-Dry Transfer 54 2.2.6.5 lmmunoblotting 55 2.2.6.6 Western Blotting for SlLAC Evaluation 56 2.2.7 Coimmunoprecipitation 57 2.2.7.1 Astrocyte Treatment 58 2.2.7.2 Sample Collection 58 2.2.8 Proximity Ligation Assay 59 2.2.8.1 Cell Treatment 6 1 2.2.8.2 Proximity Ligation Assay - Procedure 6 1 2.2.8.3 Cell Staining 62 2.2.9 Sample Preparation for Mass Spectrometry 62 2.2.9.1 Primary Astrocyte Cultures 63 2.2.9.2 Medium Collection and Protein Precipitation 64 2.2.9.3 Protein Separation by Gel Electrophoresis 64 2.2.9.4 Gel Fixation 66 2.2.9.5 In-Gel Protein Digestion 66 2.2.9.6 Peptide Extraction 6 7 2.2.9.7 Mass Spectrometry 68 2.2.9.8 Data Analysis 69 2.2.9.9 Data Treatment and Visualisation 7 1

3. Chapter l

3.1 Chapter IA 3.1 .I Introduction 3.1 .I.I lntracellular Vesicles 3.1 .I.2 Endosomal Markers 3.1 -1.3 Studying Vesicular Compartments 3.1.2 Results 3.1.2.1 Cell Type-Specific Angiogenin lnternalisation 3.1.2.2 Astrocyte Morphology 3.1.2.3 Angiogenin Sorting after lnternalisation 3.1.3 Discussion

3.2 Chapter IB 3.2.1 lntroduction 3.2.1 .I Endocytic Mechanisms 3.2.1.2 Coated Pit Formation 3.2.1.3 Vesicle Budding 3.2.1.4 Studying the Endocytic Mechanism 3.2.2 Results 3.2.2.1 Angiogenin Endocytosis is Clathrin-Dependent 3.2.2.2 Angiogenin Vesicle Budding Requires Dynamin Function 3.2.2.3 Angiogenin Endcytosis is Sodium- and Heparin- Dependent 3.2.3 Discussion

3.3 Chapter IC 3.3.1 lntroduction 3.3.1 .I Cell Surface Proteoglycans 3.3.1.2 Syndecan 4 3.3.1.3 Colocalisation Analysis 3.3.2 Results 3.3.2.1 Angiogenin Binds Syndecan 4 on Astrocytes 3.3.2.2 Suppressing Syndecan 4 Expression Inhibits Angiogenin Uptake 3.3.2.3 Syndecan 4 Can Induce Uptake of Angiogenin 3.3.2.4 Signalling Elicited by Angiogenin Binding Syndecan 4 3.3.3 Discussion 4. Chapter ll

4.1 Chapter IIA 4.1 .I lntroduction 4.1 .I.I Mass Spectrometry 4.1 .I.2 Stabel Isotope Labelling by Amino Acids in Cell Culture 4.1 .I.3 Analysis of Mass Spectra 4.1.2 Results 4.1.2.1 Incorporation of SlLAC Amino Acids 4.1.2.2 Treatment Protocol Optimisation 4.1.3 Discussion

4.2Chapter IIB 4.2.1 lntroduction 4.2.1 .I Secretory Routes 4.2.1.2 Astrocyte-Secreted Proteins 4.2.2 Results 4.2.2.1 Comparison with Published Data 4.2.2.2 Gene Ontology Analysis 4.2.2.3 KEGG Analysis of Enriched Pathways 4.2.3 Discussion

4.3Chapter IIC 4.3.1 lntroduction 4.3.1.1 Studying Astrocyte Secretion under Inflammatory Stimulation 4.3.1.2 Studying Astrocyte Stimulation under Neuronal Stimulation 4.3.2 Results 4.3.2.1 Secreted Proteins Regulated by Angiogenin 4.3.2.2 Changes in Functional Annotation with Angiogenin Treatment 4.3.2.3 Validation of Selected Proteins 4.3.3 Discussion

5, Discussion 180

5.1 Angiogenin Internalisation by Astrocytes 180 5.1 .I The Angiogenin Receptor on Astrocytes 180 5.1.2 Angiogenin in Cell Adhesion 181 5.1.3 Angiogenin Containing Vesicles May Serve Multiple Purposes 182 5.1.4 Angiogenin Escape from Vesicles 185

5.2Angiogenin Effects on Astrocytes 187 5.2.1 Angiogenin Signalling 187 5.2.2 The Astrocyte Secretome in Response to Angiogenin 188 5.2.3 Fine Tuning of Astrocyte Secretion 190 5.2.4 Angiogenin in the Brain: Signalling Directionality 193

5.3 Future Perspectives for ALS Research 196

6. Bibliography 198 Acknowledgements

I would like to thank Prof. Jochen Prehn and Dr. Philippe Marin for giving me the opportunity to perform this study at both their laboratories in Dublin and Montpellier and for their support and guidance throughout the project.

Thank you to Dr. Matthew King for all the advice and fruitful discussions and the rest of the ALS team in Dublin, Bridget, Ina, Sarah and Aine, for their help.

I would also like to thank Dr. Heiko Diissmann and Dr. Tytus Bernas for their help with the fluorescent imaging and subsequent analysis and Dr. Hans- Georg Konig and Dr. Beau Fenner for their advice.

To all the former and current members of the Department of Physiology and Medical Physics, thank you for the pleasant and friendly working atmosphere and special thanks to Ujval, Aurelien, Monika, Beatrice, Susan, Gary and Franziska for your friendship.

I am very grateful for the warm welcome by the Neurobiology and Proteomics teams in Montpellier. Serge, Edith and Oana, you have been of tremendous help performing the proteomics study. I would also like to thank Florence, Severine, Sylvie and Carine for their technical advice. Special thanks to Guillaume, Samah, Maud, Julie, Romain, Pascal, Franck and Alexander for your friendship.

Thank you to Dr. Jean-Francois Dubremetz for performing the electron microscopy and Samira for preparing the samples with me.

I would like to thank Pascal, Emmet and Iris for their corrections, they have been of great help.

I would like to express my gratitude towards my family and friends for the continuous encouragement from near and far and for diverting my attention also to other things.

Thank you to Martin, for being there for me, for your trust, encouragement and love.

Finally, I would like to thank the National Biophotonics and Imaging Platform Ireland for financially supporting this project. Summary in English

Amyotrophic Lateral Sclerosis (ALS) is a progressive, fatal neurodegenerative disease of the motor system affecting both upper and lower motoneurons. The majority of ALS cases occur sporadically, however roughly 10 % show a hereditary component. Mutations in the hypoxia- inducible factor angiogenin segregate with ALS pedigrees and the protein is expressed in motoneurons. Angiogenin shows potent neuroprotective properties in vitro and in vivo and application of recombinant human angiogenin to mixed spinal cord cultures revealed non-neuronal uptake of the protein. As the disease affects both motoneurons and non-neuronal neighbouring cells like astrocytes and microglia, a paracrine mechanism of neuroprotection has been suggested.

This study was performed to gain insights into the mechanism of angiogenin- induced neuroprotection via surrounding glial cells focusing on astrocytes. The vesicular internalisation of recombinant human angiogenin by primary astrocytes was investigated in culture using pharmacological inhibitors, cell transfection and immunocytochernistry. This revealed that angiogenin endocytosis by astrocytes is clathrin-dependent and involves dynamin for vesicle scission. Vesicle budding and trafficking do not require a functional microtubule network. A fraction of the internalised angiogenin is targeted for lysosomal degradation, while the majority remains in uncharacterised sorting endosomes. The receptor for angiogenin on astrocytes was identified to be the heparansulfate proteoglycan syndecan 4 by colocalisation studies and protein knock down.

To further elucidate the role of angiogenin in paracrine neuroprotection, modification of the astrocyte secretome was investigated in response to angiogenin treatment by quantitative mass spectrometry. Metabolic protein labelling by Stable Isotope Labelling with Amino acids in Culture (SILAC) was optimised for primary astrocytes and proteins were identified by Fourier transform tandem mass spectrometry (nano-LC-FT-MSIMS). This screen identified over 1,500 proteins in the supernatant of primary astrocytes, many viii of which are known to exhibit extracellular location like components of the Extracellular Matrix (ECM), cytokines, growth factors and growth factor binding proteins. However, proteins with established intracellular function like splicing factors and ribosomal proteins were detected, too. Quantification using the MaxQuant software showed significant regulation of over 100 proteins in response to angiogenin treatment. The most strongly regulated proteins are involved in modifying the ECM or contribute to immune responses and might be responsible for the neuroprotective effects of angiogenin. These data also suggest the involvement of surrounding immune and endothelial cells in the biological activity of angiogenin. Additionally, local transfer of factors involved in protein translation from astrocytes to surrounding cells may be affected by angiogenin.

In conclusion, this study has shed new light on the role of angiogenin in the complex cellular interplay in the Central Nervous System (CNS) focusing on astrocytes and how they interact with neighbouring cells. Further studies will be necessary to elucidate the functional signalling outcome of angiogenin focusing on its neuroprotective activities, in particular regarding ALS pathology with the aim of finding new therapies for this fatal disease. Resume en Fran~ais

La sclerose laterale amyotrophique (SLA) est une maladie neurodegenerative affectant specifiquement les motoneurones de la moelle epiniere et de I'encephale et qui presente une composante hereditaire dans 10% des cas. Un des genes impliques code pour I'angiogenine, une proteine induite par I'hypoxie exprimee par les motoneurones et qui exerce de puissants effets neuroprotecteurs in vitro et in vivo. L'application d'angiogenine sur des cultures mixtes de neurones et de cellules gliales de moelle epiniere s'ensuit d'une recapture astrocytaire de I'angiogenine. Du fait de I'implication des cellules non-neuronales comme les astrocytes dans la SLA, un mecanisme paracrine a ete envisage pour expliquer les effets neuroprotecteurs de I'angiogenine.

Afin d'elucider les mecanismes impliques dans ces effets neuroprotecteurs, j'ai dans un premier temps etudie le trafic de I'angiogenine recaptee par les astrocytes. J'ai demontre que I'endocytose de I'angiogenine dependait de la clathrine et de la dynamine et que son trafic vesiculaire etait independant du reseau des microtubules. De plus, une fraction de I'angiogenine internalisee est adressee dans les lysosornes alors que la majorite de celle-ci reste localisee dans un compartiment endosomal. Des etudes de co-localisation et d'invalidation par ARN interference ont permis d'etablir que le recepteur astrocytaire de I'angiogenine etait le syndecan 4, un proteoglycan a heparane sulfate.

J'ai dans un deuxieme temps analyse les modifications du secretome astrocytaire par I'angiogenine, grice a une approche proteomique quantitative associant la technologie SlLAC (Stable Isotope Labelling of Amino acids in culture), I'identification et la quantification des proteines par spectrometrie de masse a transformee de Fourier. Cette approche a permis d'identifier plus de 1500 proteines dans le surnageant de cultures astrocytaires. Un grand nombre d'entre elles, incluant des proteines de la matrice extracellulaire, des cytokines, des facteurs de croissance et des proteines associees a ceux-ci, presente une localisation extracellulaire. Toutefois, des proteines connues pour &re localisees dans les compartiments intracellulaires, comme des facteurs d'epissage et des proteines ribosomales, ont egalement ete identifiees dans le surnageant astrocytaire. La quantification relative des proteines grgce au logiciel MaxQuant a mis en evidence une expression differentielle de plus de 100 proteines dans le surnageant d'astrocytes traitees ou non a I'angiogenine. Les proteines presentant les differentiels les plus importants sont connues pour modifier la matrice extracellulaire ou participer a la reponse immunitaire et pourraient contribuer a I'effet neuroprotecteur de I'angiogenine. Les resultats de ce crible suggerent egalement I'implication de cellules immunitaires et endotheliales dans les effets biologiques de I'angiogenine et que celle-ci puisse affecter le transfert de facteurs impliques dans la traduction des astrocytes aux cellules environnantes.

En conclusion, cette etude a apporte un nouvel eclairage sur le r61e de I'angiogenine dans les interactions cellulaires dans le systeme nerveux central (SNC), notamment entre astrocytes et cellules environnantes. Des etudes complementaires seront necessaires pour elucider les mecanismes neuroprotecteurs declenches par I'angiogenine, en particulier dans la SLA, dans la perspective d'identifier de nouvelles approches therapeutiques dans cette maladie neurodegenerative incurable. Publications and Presentations

Journal article

Alexandra Skorupa, Matthew A King, Bridget Breen, Dairin Kieran, Caoimhin G. Concannon, Philippe Marin, Jochen HM Prehn. Motoneurons secrete angiogenin to induce RNA cleavage in astroglia. Manuscript in preparation

Posters

Alexandra Skorupa, Serge Urbach, Matthew A King, Severine Chaumont- Dubel, Jochen HM Prehn, Philippe Marin. Quantitative proteomic analysis reveals modification of astrocyte secretome upon angiogenin treatment. Proteomic Forum Berlin 201 1

Alexandra Skorupa, Matthew A King, Heiko Duessmann, Philippe Marin, Jochen HM Prehn. Angiogenin uptake into astrocytes. Biophotonics & Imaging Graduate Summer School (BIGSS) 201 0

Matthew A King, Alexandra Skorupa, Bridget Breen, Jochen HM Prehn. Angiogenin signalling in the spinal cord. 2oth International Symposium on ALSlMN 2009

Alexandra Skorupa, Matthew A King, Philippe Marin, Jochen HM Prehn. Angiogenin uptake into astrocytes. International conference of the Society for Neuroscience (SfN) 2009 Abbreviations

aFGF acidic Fibroblast growth factor Ang Angiogenin ALS Amyotrophic lateral sclerosis AMPA a-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate APS Ammonium persulfate BCA Bicinchoninic acid bFGF basic Fibroblast growth factor BSA Bovine serum albumin CAMP cyclic AMP CID Collision-induced dissociation CSF Cerebrospinal fluid CNS Central nervous system Col IV Collagen IV DAP l 4',6 diamidino-2-phenylindole dH20 deionised water DIV Days in vitro DMEM Dubecco's modified Eagle medium DNA Deoxyribonucleic acid EAAT2 Excitatory amino acid transporter 2 ECL Enhanced chemiluminescence ECM Extracellular matrix ER Endoplasmic reticulum Erkll2 Extracellular signal-regulated kinase 112 EtOH Ethanol FAK Focal adhesions kinase FBS Fetal bovine serum FDR False discovery rate FTD Frontotemporal dementia Fus Fused in sarcoma GDNF Glial cell line-derived neurotrophic factor GEEC GPI-AP-enriched early endosomal compartments GFAP Glial fibrillary acidic protein GO Gene ontology GPCR G-protein-coupled receptor GPI Glycosylphosphatidylinositol H4 Histone H4 HRP Horse radish peroxidase lLlra Interleukin-I receptor antagonist

1 p3 lnositoltriphosphate JNK c-Jun N-terminal kinase KEGG Kyoto encyclopedia of genes and genomes LB Luria Burtani LTQ Linear trap quadrupole LTR LysoTracker Red MAP Mitogen-activated protein MPCD Methyl-P-cyclodextrin MDC Monodansylcadaverine MGI Mouse genome informatics MMP3 Matrix metalloproteinase-3 mRNA messenger RNA NB Neurobasal NeuN Neuronal Nuclei NGF Nerve growth factor NO Nitric oxide P2 Postnatal day 2 PAGE Polyacrylamide gel electrophoresis PBS Phosphate buffered saline PEP Posterior error probability PFA Paraformaldehyde PI Phosphatidylinositol PI 4,5BP Phosphatidylinositol 4,5-bisphosphate PKA Protein kinase A PKB Protein kinase B PKCa Protein kinase C alpha Plau Urokinase-type plasminogen activator

xiv PLC Phospholipase C PIS PenicillinlStreptomycin Q L-Glutamine rhAng recombinant human Angiogenin RNA Ri bonucleic acid RNase Ribonuclease rRNA ribosomal RNA RTK Receptor tyrosine kinase RT Room temperature shRNA short hairpin RNA SILAC Stable isotope labelling by amino acids in cell culture siRNA small interfering RNA snRNA small nuclear RNA SOD1 Superoxide dismutase 1 Srsfl SerinelArginine-rich splicing factor 1 Syn4 Syndecan 4 TCA Trichloroacetic acid TDP-43 Transactive response DNA binding protein TEMED Tetramethylethylenediamine TGF-p Transforming growth factor-p Tgfbi Transforming growth factor-P-induced protein ig-h3 tiRNA tRNA-derived stress-induced RNAs TNFa Tumour necrosis factor a tRNA transfer RNA VAPB Vesicle-associated membrane protein-associated protein B VEGF Vascular endothelial growth factor

1. Introduction

1.I Amyotrophic Lateral Sclerosis

1.I .I Aetiology and Symptoms

Amyotrophic Lateral Sclerosis (ALS) is a fatal, progressive neurodegenerative disorder, characterised by the relatively selective loss of both upper and lower motoneurons (Cleveland and Rothstein, 2001) and was first described by the French neurobiologist Jean-Martin Charcot (Charcot and Joffroy, 1869). Upper motoneurons are the cortical neurons emanating from the motor cortex to the brain stem (corticobulbar neurons) or spinal cord forming the pyramidal tract, where they contact lower motoneurons. These neurons generate the signal for muscle contraction, thus their axons leave the ventral horn of the spinal cord to connect to the corresponding muscles in the periphery of the body. Accordingly, the disease is characterised by symptoms caused by degeneration of the motoneurons involved. For upper motoneurons these are (amongst others): hyperreflexia, increased muscle tone and weakness in topographical representation. Lower motoneuron involvement is characterised by weakness, hyporeflexia, muscle cramps and fasciculations, thus early presentation is variable. The disease initially affects extremities (limb-onset) or more rarely involves motor functions coordinated by the brain stem, which causes so-called bulbar symptoms (bulbar-onset) such as difficulties speaking or swallowing. Muscle atrophy and weakness spread throughout the body during disease progression, yet oculomotor neurons are only rarely affected in patients with very long disease course. Patients eventually die from respiratory failure. ALS pathology has been linked with frontotemporal dementia (FTD), a clinical syndrome resulting from degeneration of the frontal lobe of the cortex that profoundly alters personality and social conduct (reviewed by Donkervoort and Siddique, 1993; and Boillee et al., 2006a; and Kiernan et al., 201 1).

Every year the number of newly diagnosed individuals with ALS is between one and three per 100,000 with men having a higher incidence of disease than women. Due to the short survival period of two to five years following diagnosis, the prevalence is not much higher at four to eight per 100,000. Even though for most ALS patients the specific aetiology is unknown, roughly 10 % of ALS cases show a hereditary component. Sporadic and familial ALS differ in the mean age of disease onset, which is 56 years for sporadic ALS and only 46 years for familial cases of the disease. Possibly, environmental factors also play a role in ALS pathogenesis. Mutations at the superoxide dismutase 1 (SOD?) locus represent the most common genetic determinant

in about 20 Oh of all familial ALS cases (Rosen, 1993) and 5 % of sporadic ALS patients. Currently, thirteen genes and loci which contribute to ALS pathology have been indentified (reviewed by Donkervoort and Siddique, 1993; and Boillee et al., 2006a; and Kiernan et al., 201 1).

1. I.2 Familial ALS Mutations

The gene encoding SOD1 is found on chromosome 21 and the translated protein is a ubiquitously expressed enzyme of 153 amino acids. Its function is to convert free superoxide radicals to hydrogen peroxide for further detoxification by glutathione peroxidase or catalase to water and oxygen. Two additional SOD enzymes are found in mammals and should be distinguished: a mitochondria1 form called SOD2 and the extracellular SOD3. Loss of SOD1 detoxifying activity was initially hypothesised as a cause of ALS pathology. However, further studies performed on rodent models revealed that the patho-physiological mechanism involved in ALS is much more complex and involves toxic gain of function of the protein (see below; reviewed by Boillee et al., 2006a; and Bento-Abreu et al., 2010). Following identification of mutations in the gene encoding SOD1 in familial cases of ALS, several other mutations have been found and characterised:

The typical clinical phenotype is additionally caused by mutations in the genes encoding transactive response DNA binding protein 43 (TDP-43), Fused in sarcoma (Fus), angiogenin (Ang) and optineurin. TDP-43 was originally found in intraneuronal inclusions characterising both ALS and FTD (Arai et al., 2006) and only later observed to be mutated in familial and sporadic ALS patients (Sreedharan et al., 2008; Yokoseki et al., 2008). The two proteins TDP-43 and Fus are multifunctional proteins that contribute to several steps in gene regulation and protein synthesis such as gene transcription as well as RNA splicing, transport and translation. Both proteins mainly localise to the nucleus and have been found to shuttle between nucleus and cytoplasm under physiological conditions, but they redistribute to the cytoplasm if mutated. Angiogenin mutations were found to be associated with both familial and sporadic ALS cases (Greenway et al., 2006). The secreted protein displays ribonucleolytic activity encoded by a hypoxia- responsive gene and has also been implicated in RNA metabolism (see pg. 15). These findings have initiated many studies investigating the role of RNA processing in motoneuron degeneration (reviewed by Baumer et al., 2010). ALS-associated mutations in optineurin affect the protein's cytoplasmic distribution and the inhibition of tumour necrosis factor a (TNFa)- induced activation of nuclear factor KB is abolished (reviewed by Bento- Abreu et al., 2010; Maruyama et al., 2010; reviewed by Kiernan et al., 201 1).

The vesicle-associated membrane protein-associated protein B (VAPB) was found to be mutated in Brazilian patients (Nishimura et al., 2004). VAPB is ubiquitously expressed and is normally involved in the unfolded protein response in the endoplasmic reticulum (ER), which is triggered by accumulation of unfolded proteins. Mutant VAPB has also been shown to misfold and aggregate. Another gene associated with both sporadic and familial ALS cases is factor-induced gene 4 (Chow et al., 2009). The encoded phosphatase regulates phosphatidylinositol 3,5-bisphosphate levels and is involved in autophagy. The gene encoding the dynactin subunit p150Glued was found to be mutated in atypical sporadic ALS. Dynactin is required for cytoplasmic dynamin-driven retrograde transport of vesicles and organelles along the microtubule cytoskeleton. Mutations in alsin, encoding a guanine nucleotide exchange factor for the small GTPases Rab 5 and Rac 1, and senataxin, which encodes a probable helicase likely involved in RNA maturation, cause motoneuron diseases closely resembling ALS (reviewed by Boillee et al., 2006a; and Bento-Abreu et al., 2010).

1.I .3 SODl Mutant Toxicity

The identification of SODl in familial ALS cases led to generation of transgenic mice and rats expressing different mutated human SODl genes under the control of the endogenous promoter. These transgenic animals all develop a lethal disease resembling human ALS, however the molecular observations made on the basis of these models are highly variable. The animal models have been studied extensively in an attempt to find potential therapies to cure or at least ameliorate the disease. However, several compounds that showed positive effects in the disease model have proven to be ineffective in studies on humans, thus it has been questioned whether it is an appropriate model organism to use. To date however, the SOD1 animal model has been a valuable tool to study ALS pathology and to characterise the disease course (reviewed by Turner and Talbot, 2008; and Bento-Abreu et al., 201 0).

Mutations in SODl have been observed to cause dysfunction of the axon as one of the earliest manifestations, which leads to retraction of the motoaxon from the neuromuscular junction leaving the muscle denervated (Fischer et al., 2004), thus preserving the cell body is not sufficient to ameliorate the clinical disease phenotype (Gould et al., 2006; Dewil et al., 2007). Neither SODI-deficient mice (Reaume et al., 1996) nor mice expressing high levels of human wildtype SODl (Gurney et al., 1994; Wong et al., 1995) develop motoneuron disease and wildtype SODl expression is maintained in the animal model, but disease severity has been correlated with SODl mutant expression levels (Alexander et al., 2004). These observations led to the 4 conclusion that neuron toxicity is caused by a toxic gain-of-function not just a loss-of-function. Protein aggregates are commonly observed in neurodegenerative diseases such as ALS. Mutant SOD1 has been shown to be prone to aggregation especially in motoneurons (Durham et al., 1997), as it does not fold properly and does not assemble normally into dimers during protein synthesis at the endoplasmic reticulum (Figure 1) (reviewed by Nordlund and Oliveberg, 2008). This misfolding may cause ER stress by triggering the unfolded protein response, which leads to upregulation of chaperone protein synthesis to refold newly synthesised proteins. If this fails, the proteins are exported to the cytoplasm and targeted for degradation by the proteasome following ubiquitination. Motoneurons have a high threshold for inducing the unfolded protein response (Batulan et al., 2003), possibly making them particularly vulnerable. Mutant SODl accumulates in the ER (Nishitoh et al., 2008) and has also been detected in cytoplasmic inclusions (Jonsson et al., 2006). Aggregates may also contain other components of the cytoplasm with essential functions in cell physiology, thus severely disrupting homeostasis. Mitochondria often show abnormalities in aging-related neurodegenerative disorders and mutant SODl accumulates at the outer mitochondrial membrane (Liu et al., 2004; Vande Velde et al., 2008), while wildtype SODl is normally found in the intermembrane space. This redistribution at mitochondria could affect protein import, transport of the organelle along the cytoskeleton or mitochondrial fusion and fission. Recently, mutant SODl has been reported to be secreted from motoneurons (Urushitani et al., 2006) with toxic consequences for the motoneurons themselves (reviewed by Boillee et al., 2006a; and Bento-Abreu et al., 2010; and Kawamata and Manfredi, 2010; Zhao et al., 2010). Mutation Figure 1: SODl mutant toxicity. Mutant SODl protein can coaggregate with other essential proteins, inhibit proper function of the proteasome or deplete the cell of chaperones. At the outer membrane of mitochondria, mutant SOD1 accumulates with possible consequences mitochondria1 Lass of Clagglng of Deplellan of D sfunction of function (Boillee et al., proleamme chaperones rnrtochondna 2006a). r;:z;:fl with andlor other through rnisfolded organelles w-aggregation proteins

Evidence is accumulating that ALS is not just a disease affecting motoneurons, but surrounding glial cells like astrocytes and microglia contribute to motoneuron toxicity (see pg. 10). This was first studied using SODl mutantlwildtype chimeric animals, which do not express the mutant protein in all cells: If individual motoneurons express mutant SODl even at high levels, they do not degenerate. In SODl mutant mice not expressing the mutant protein in motoneurons (by means of expressing Cre recombinase under the motoneuron-specific Islet-I promoter), both disease onset and early progression were slowed down (Boillee et al., 2006b). Thus, mutant SODl protein must also affect surrounding cell types. Accordingly, diminishing the SODl mutant protein from microglia (Cre recombinase under microglia-specific CDl Ib promoter) did not affect disease onset but drastically slowed disease progression. Likewise, disease onset remained unchanged but progression sharply slowed with abolishing SODl mutant protein expression selectively in astrocytes (Cre recombinase under the astrocyte-specific GFAP promoter; Yamanaka et al., 2008). This effect was partly explained by concomitant reduction of microglial activation, indicating interaction of the two cell types (reviewed by Boillee et al., 2006a; and llieva et al., 2009; and Bento-Abreu et al., 2010). 1.I .4 Neurodegeneration in ALS

Studying ALS pathology in patients, animal models and cell culture systems led to the identification of several possible mechanisms involved in neurodegeneration causing ALS pathology (summarised in Figure 2; highlighted in the text). However, it remains elusive which of the observed effects are cause or consequence of each other. Furthermore, there is ongoing controversy as to whether the disease starts mainly by affecting upper motoneurons, which cause degeneration of lower motoneurons by glutamate excitotoxicity. Alternatively, a motoneuron-specific trophic factor could be lacking at the neuromuscular junction, leading to degeneration of initially lower motoneurons followed by upper motoneurons (reviewed by Kiernan et al., 201 1).

As mentioned above, protein aggregates are often observed in ALS pathology, but their contribution to ALS pathogenesis is unresolved. Within the axon itself, misaccumulation of neurofilaments leads to alterations in axonal structure. These changes could affect axonal transport of organelles, contributing to neurodegeneration. This finding is supported by the identification of mutations in the dynactin subunit required for vesicle transport mentioned before (see pg. 2). Furthermore, recent genome-wide association studies have identified additional factors required for axonal structure and vesicle release (reviewed by Boillee et al., 2006a; and Goodall and Morrison, 2006; and Bento-Abreu et al., 2010).

Despite the finding that SOD1 mutant protein pathology cannot be explained on the basis of a simple loss-of-function, oxidative damage to DNA, lipids and proteins has been reported in ALS patients. Interestingly, the glutamate transporter EAAT2 (excitatory amino acid transporter 2) is frequently damaged by oxidation (reviewed by Trotti et al., 1998), linking ALS pathology to glutamate excitotoxicity. Some ALS patients have elevated levels of glutamate in their cerebrospinal fluid (CSF; Shaw et al., 1995; Spreux- Varoquaux et al., 2002) and motoneurons are very sensitive to excitotoxicity as they express high numbers of calcium-permeable glutamate receptors (Carried0 et al., 1996; Van Den Bosch et al., 2000; Van Damme et al., 2002). 7 However, motoneurons have only limited calcium buffering capacity due to low expression levels of the required calcium-buffering proteins (Alexianu et al., 1994). Additionally, faulty editing of the messenger RNA encoding one of the glutamate receptor subunits (GluR2) in sporadic ALS patients may further increase calcium permeability and thus motoneuron vulnerability (Kawahara et al., 2004). The observed defect in messenger RNA editing also establishes a link between the pathomechanism causing ALS and RNA metabolism indicated above (see pg. 2) by the identification of variants of RNA editing proteins (TDP-43, Fus) in ALS patients (reviewed by Goodall and Morrison, 2006; and Bento-Abreu et al., 2010; and Kawamata and Manfredi, 201 0).

Abnormal mitochondrial morphology has been observed in neurodegeneration, in ALS patients mitochondria contain elevated calcium levels (Siklos et al., 1996) and display decreased activity of respiratory chain complexes (Borthwick et al., 1999; Wiedemann et al., 2002) indicating mitochondrial dysfunction also in ALS pathology. SODl mutant protein has been suggested to contribute to the observed dysfunction, but the exact mechanism remains elusive. Mitochondria play an important role in buffering calcium, especially in motoneurons under excitotoxic stress. They also exert crucial functions in apoptosis, programmed cell death, by releasing cytochrome c and triggering the caspase cascade. ALS patients and SODl mutant mice show decreased expression of the antiapoptotic protein Bcl-2 in spinal cord motoneurons (Mu et al., 1996; Ekegren et al., 1999; Gonzalez de Aguilar et al., 2000; Vukosavic et al., 2000) and Bcl-2 forms aggregates in mitochondria with mutant SODl protein (Pasinelli et al., 2004; Pedrini et al., 2010). In spinal cord tissue from ALS patients the caspases 1 and 9 are activated (Li et al., 2000; lnoue et al., 2003), indicating a role of apoptosis in neurodegeneration (reviewed by Boillee et al., 2006a; and Goodall and Morrison, 2006; and Bento-Abreu et al., 2010; and Kawamata and Manfredi, 201 0).

Analysis of CSF and spinal cord proteins in ALS patients showed decreased levels of vascular endothelial growth factor (VEGF; Devos et al., 2004; Brockington et al., 2006), a growth factor upregulated in response to hypoxia, establishing a link between ALS and the hypoxia-induced response. Earlier observations had shown that mice bearing a mutation in the hypoxia- response element of the VEGF promoter sequence develop an ALS-like motoneuron disorder (Oosthuyse et al., 2001). More recently, missense mutations in the hypoxia-sensitive factor angiogenin have been associated with ALS (Greenway et al., 2006), further underlining the importance of hypoxia-regulated processes with regard to the pathomechansim of ALS. Additionally, this also emphasises the contribution of neighbouring cells, as it establishes a link with the vascular system (reviewed by Goodall and Morrison, 2006; and Bento-Abreu et al., 2010).

Figure 2: Neurodegeneration in ALS is caused by cellular and molecular mechanisms. Within neurons, glutamate excitotoxicity, oxidative stress by free radical generation, cytoplasmic aggregates (TDP-43, Fus, SODA), mitochondria1 dysfunction and disruption of axonal transport due to neurofilament aggregates contribute to degeneration. Surrounding activated microglia secrete proinflammatory cytokines causing further toxicity. Astrocytes become impaired in their supportive functions such as glutamate clearance additionally aggravating neuron toxicity (Kiernan et al., 201 1 ). 1.I .5 Cellular Players in ALS Pathology

In addition to the mechanisms summarised in the previous section, which contribute to neurodegeneration in ALS within the motoneurons themselves, evidence is accumulating with regard to the role of neighbouring cells in motoneuron death as indicated above (see pg. 4 and Figure 2). Lower motoneurons specifically contact muscle cells via the neuromuscular junction. In the brain however, neurons are surrounded by microglia, cells of hematopoietic origin, which constitute major part of the immune system in the central nervous system (CNS). Additionally, astrocytes are found in close association with neurons, which form a scaffold for neurons but also contribute majorly to brain homeostasis and support neuronal function. All of the cell types mentioned above have been implicated in the pathomechanism of ALS and will be discussed briefly below in an attempt to highlight the importance of functional interplay between all cells involved. The role of astrocytes in ALS will be discussed in the next section (see pg. 11).

Microglia continuously monitor the CNS looking for pathogens or injury in a resting state. A neuroinflammatory response is triggered upon injury to activate microglia so they can phagocytose pathogens, present antigens and secrete reactive oxygen species, cytokines and growth factors. Two different activation states have been observed: Cytotoxic MI microglia secrete reactive oxygen species and proinflammatory cytokines (TNFa and interleukin-1 P). Proinflammatory responses are blocked by M2 microglia as they release high levels of anti-inflammatory cytokines (interleukins-4 and-I 0) as well as trophic factors (Insulin-like growth factor). Microglial activation majorly contributes to ALS pathology, yet it may promote motoneuron injury or protection. In ALS model mice, anti-inflammatory M2 microglia appear first in ALS pathology and only later more of the MI type microglia are observed releasing proinflammatory cytokines. Furthermore, in ALS post mortem tissue and model mice, the levels of cyclooxygenase 2 are elevated. This enzyme, which is responsible for production of proinflammatory prostaglandins, is expressed by microglia, astrocytes and neurons. Prostaglandins act by stimulating the production of proinflammatory cytokines, which further underlines the importance of inflammatory responses in ALS pathology (reviewed by Boillee et al., 2006a; and Goodall and Morrison, 2006; and Henkel et al., 2009; and Vargas and Johnson, 2010; and Philips and Robberecht, 201 1).

Despite the observation that axonal degeneration is one of the earliest signs of ALS pathology, the role of muscle cells in disease progression is unclear. Mitochondria1 defects are present in muscle tissue and decreased respiratory chain activity in skeletal muscle of ALS patients has been reported. Other studies point towards elevated muscle metabolic activity in ALS pathology and increased oxidative stress in myoblasts derived from ALS patients. Interestingly, physical exercise can be beneficial in animal models, however this could not be confirmed in studies on ALS patients. Mutant SOD1 expression exclusively in skeletal muscle did result in progressive muscle weakness, indicating a role of muscle cells in ALS pathology (reviewed by Boillee et al., 2006a; and llieva et al., 2009; and Kawamata and Manfredi, 2010; and Murray et al., 2010).

1.I .6 Astrocytes Secrete Toxic and Protective Factors

Astrocytes perform important functions in CNS physiology by organising the brain into distinct subunits, which are the spatial territories covered by any individual astrocyte with its many processes. These territories show only little overlap and every astrocyte normally contacts at least one blood vessel with its end feet. Therefore, astrocytes are ideally positioned to provide trophic and metabolic support for neurons and to regulate cerebral blood flow to meet the needs of neurons (reviewed by Gordon et al., 2007; and Ben Achour and Pascual, 2010; and Vargas and Johnson, 2010).

Astrocytes more directly protect motoneurons, by secreting neurotrophic factors, aiding extracellular glutamate clearance and supplying antioxidant molecules (Figure 3). Brain-derived neurotrophic factor, glial cell line-derived

11 neurotrophic factor (GDNF) and ciliary neurotrophic factor have been shown to be able to rescue motoneurons both in vitro and in vivo. Additionally, VEGF is secreted by astrocytes and is neurotrophic for motoneurons. Enhancing the neuroprotective properties of astrocytes can be used to find novel therapeutic strategies (reviewed by Ekestern, 2004; and Van Den Bosch and Robberecht, 2008; and Philips and Robberecht, 201 1).

Neuroprotection Neurodegeneration

Death receptor ligands BDNF, CNTF, VEGF NGF, FaslFasL, TNFa

Glutamate transport Excitotoxicity EAAT2 EAAT2 loss

Antioxidant activity Oxidative stress Glutathione iNOS. COX2

Figure 3: Astrocytes can serve both neuroprotective and neurotoxic roles in ALS. Neuroprotection can be mediated via the secretion of trophic factors, by aiding glutamate clearance and providing antioxidants, all of which can be targeted for therapy. Astrocytes can enhance neurodegeneration by aggravating glutamate excitotoxicity, increasing oxidative stress and expressing death receptor ligands (modified from Vargas and Johnson, 2010).

In ALS pathology, astrogliosis surrounding upper and lower motoneurons is one of the most prominent characteristics and is now considered to be directly involved in motoneuron degeneration, not just a secondary change. Reactive astrocytes express higher levels of the astroglial marker glial fibrillary acidic protein (GFAP) and the calcium-binding protein SIOOP along with inflammatory markers like cyclooxygenase-2, inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase. Experimentally damaging motoneurons induces increased expression of diffusible inflammatory molecules, which can trigger glial responses similar to those observed in ALS models (Olsson et al., 1989; Huber et al., 1997). Altered protein secretion by astrocytes in ALS may either lead to impaired 12 neurotrophic support of motoneurons or alternatively, astrocytes may release toxic factors. In SOD1 mutant animals, astrocytes have been shown to secrete mutant SODl protein, which can activate microglia and induce motoneuron degeneration. Mutant SODI-expressing astrocytes can induce cell death in wildtype motoneurons (Di Giorgio et al., 2007), probably by secreting factors toxic to motoneurons as shown for mouse primary spinal cord astrocytes expressing mutant SODl (Nagai et al., 2007). The same study confirmed that the secreted factor was selectively toxic to motoneurons and not secreted by other cells like microglia, fibroblasts, cortical neurons or myocytes expressing SODl mutant protein. Taken together, astrocytes can secrete both protective and toxic molecules depending on the cellular environment (reviewed by Pehar et al., 2005; and Van Den Bosch and Robberecht, 2008; and Vargas and Johnson, 2010).

I.I .7 Astrocytes Specifically Interact with Motoneurons

Besides the secretion of neurotrophic factors by astrocytes, they also contribute to synaptic transmission as part of what is now referred to as the tripartite synapse, including the astrocyte process and both pre- and postsynaptic nerve terminals (a concept introduced by Araque et al., 1999). As mentioned above, astrocytes contribute to extracellular glutamate clearance by expressing the glutamate transporter EAAT2 to remove extracellular glutamate. The expression level of EAAT2 is regulated by presynaptic neurons and neurodegenerative injury results in reduced EAAT2 expression. Interestingly, the density of glutamate transporters in spinal cords of ALS patients was reported to be reduced (Shaw et al., 1994). This observation was later observed to be caused by loss of the astroglial glutamate transporter EAAT2 in both the motor cortex and the spinal cord of ALS patients (Rothstein et al., 1995), which could lead to accumulation of excitotoxic levels of extracellular glutamate in ALS (reviewed by Van Den Bosch and Robberecht, 2008; and Vargas and Johnson, 2010; and Philips and Robberecht, 2011). Recently, it has been observed that astrocytes regulate the expression of a non-calcium permeable subunit of the glutamate receptor (GluR2) on motoneurons by secreting an unknown factor, thus protecting them from glutamate-induced excitotoxicity (Van Damme et al., 2007). Astrocytes expressing mutant SOD1 protein have lost this ability to protect motoneurons (reviewed by Van Den Bosch and Robberecht, 2008; and Bento-Abreu et al., 2010; and Vargas and Johnson, 2010; and Philips and Robberecht, 201 1).

In addition to the mechanisms elucidated above, reactive astrocytes have been suggested as a source of nitric oxide (NO), which can cause mitochondria1 dysfunction and cell death in motoneurons. Furthermore, motoneurons express high levels of acidic fibroblast growth factor (aFGF), which is released in response to cell injury or stress and can activate surrounding astrocytes. Following activation by aFGF, astrocytes express antioxidant enzymes to synthesise and release of neuroprotective glutathione which inactivates NO. Additionally, activated astrocytes secrete nerve growth factor (NGF), a modulator of neuronal survival especially during CNS development. Adult motoneurons do not normally express the NGF receptor p75, but it is expressed by motoneurons of ALS patients. Thus, NGF can trigger motoneuron apoptosis depending on oxidative stress levels due to NO. Cell death receptor signalling between motoneurons and either astrocytes or microglia via both FaslFas ligand and TNFa may contribute to neurodegeneration based on observations in SOD1 mutant mice, as their motoneurons are more sensitive to Fas- or NO-triggered cell death (Raoul et al., 2002; reviewed by Pehar et al., 2005; and Vargas and Johnson, 2010; and Philips and Robberecht, 201 1).

In conclusion, astrocytes influence motoneuron viability via a plethora of mechanisms by directly interacting with motoneurons or by modulating the extracellular environment. However, knowledge about neuroprotective astrocyte-secreted proteins is still limited (see pg. 149). 1.2 Angiogenin

1.2.1 Discovery

Angiogenin was the first protein derived from a human tumour cell line with in vivo angiogenic activity. The protein was purified from serum-free supernatants of the human adenocarcinoma cell line HT-29 and its angiogenic activity confirmed on the chick chorioallantoic membrane and the rabbit cornea (Fett et al., 1985). The same group published the amino acid and cDNA sequences of the isolated protein the same year (Kurachi et al., 1985; Strydom et al., 1985). It was found to be a 14.4 kDa protein of 123 amino acids, which displays 35 % homology with pancreatic ribonuclease A (RNase A) and of the remaining residues many are conservatively replaced (Strydom et al., 1985). Sequencing the cDNA with the aid of the method developed by Maxam and Gilbert (1980) showed that the angiogenin gene does not contain any intronic sequences, but is expressed as a precursor protein with a signal peptide for secretion (Kurachi et al., 1985). There is only one functional angiogenin gene in the human genome, whereas there are six in the mouse (reviewed by Strydom, 1998) and only mouse Ang-I displays angiogenic activity (Bond and Vallee, 1990). Following its discovery, the human angiogenin gene was found to be located on chromosome I4 (Weremowicz et al., 1990).

I.2.2 Molecular Properties

Angiogenins are very basic proteins even when compared to other RNases, which may be important for their biological activity, as this allows for binding of polynucleotides and proteoglycans (reviewed by Strydom, 1998). The active, folded protein is kidney-shaped and contains three disulfide bridges, unlike other RNases which contain four disulfide bonds. The three 15 dimensional structure reveals the ribonucleolytic active site as well as a putative receptor-binding site (Figure 4). The active site of RNases can be subdivided into functional subsites and the actual active sites of RNases and angiogenin display great sequence homology. Nevertheless, major differences are evident in the crystal structures of the two proteins. RNase activity depends on a pyrimidine-binding site, which is less accessible due to the Glutaminel17 residue majorly changing angiogenin's secondary structure and therefore its enzymatic activity (Acharya et al., 1994; Russo et al., 1994; reviewed by Vallee and Riordan, 1997; and Adams and Subramanian, 1999).

Figure 4: Polypeptide fold of angiogenin (left) in comparison with RNaseA (right). Both proteins display the same major structural components and the folded proteins resemble the shape of kidneys. The molecule's core is formed by two twisted P-strands (B3-B4 and B5-B6), which together with two further strands either side (B1 and B2) determine the shape of the major sheet structure. The structure is completed by short helices and loop structures. The greatest difference in structure between the two molecules exists in the loops or at the N- and C-termini (Acharya et al., 1994).

The receptor binding site comprises amino acid residues 58- 70 and Asparagineqo9,which are found on adjacent loops and differ strikingly from the other RNase family members. This confirms that the surface regions are responsible for the recognition of distinct ligands: RNases bind RNA purines, while angiogenin binds a cell surface receptor (Acharya et al., 1994). Several point mutations in the angiogenin protein have been shown to greatly increase its RNase activity, but similar mutations have opposite effects in other RNases (Acharya et al., 1994; Russo et al., 1994).

1.2.3 Angiogenin Internalisation

Angiogenin has been shown to stimulate proliferation of human endothelial cells in sparse cultures (Hu et al., 1997) and modulate mitogenic effects on different cell types (Heath et al., 1989). Several proteins binding angiogenin have been identified (reviewed by Strydom, 1998), but only one potential cell surface receptor on endothelial cells has been detected (Hu et al., 1997). This 170 kDa cell surface receptor was identified by isolating biotinylated membrane molecules from endothelial cells by avidin-affinity chromatography and applying these to an angiogenin-sepharose column to catch angiogenin- binding proteins. Furthermore, angiogenin has been found to bind cell surface actin on endothelial cells and this interaction was shown to be involved in its angiogenic activity (Hu et al., 1993). The actin-angiogenin complex was originally released from the cell surface by adding heparansulfates, which dissociates mainly proteoglycans from the plasma membrane (Hu et al., 1991).

The contribution of the two receptors could be explained as follows: Angiogenin binds endothelial cell-associated actin thereby stimulating the degradation of the extracellular matrix by activating tissue-type plasminogen activator to produce plasmin (Hu et al., 1994). Upon endothelial cell migration, their density is reduced and the 170 kDa receptor is upregulated thus angiogenin can stimulate proliferation (Hu et al., 1997; reviewed by Gao and Xu, 2008).

Receptor binding triggers internalisation and nuclear translocation by endothelial, smooth muscle and tumour cells (Moroianu and Riordan, 1994a; Xu et al., 2001; Tsuji et al., 2005). Angiogenin internalisation by endothelial cells cultured at low density was observed within 2 min of administration and was saturated at 15 min (Hu et al., 2000). This mechanism of uptake was inhibited by addition of exogenous actin or heparin and heparinase treatment (Moroianu and Riordan, 1994a). Furthermore, internalisation and nuclear translocation of angiogenin into human umbilical artery endothelial cells does not depend on microtubules or a functional lysosomal compartment (Li et al., 1997) and in cultured aortic smooth muscle cells it is internalised and processed to smaller angiogenin fragments after 24 and 48 h (Hatzi et al., 2000).

A nucleolar targeting signal was identified in angiogenin (Moroianu and Riordan, 1994b), however translocation of angiogenin to the nucleus is seemingly independent of classic nuclear import mechanisms, thus it has been suggested it may freely diffuse into the nucleus due to its small size (Lixin et al., 2001). Once inside the nucleus, angiogenin has been shown to bind DNA (Hu et al., 2000) and to stimulate rRNA (ribosomal RNA) production (Xu et al., 2002). Nuclear angiogenin itself stimulates endothelial cell proliferation and inhibiting angiogenin translocation to the nucleus abolishes its angiogenic activity (Moroianu and Riordan, 1994a; Hu, 1998). Stimulation of rRNA transcription in endothelial cells is a common feature of angiogenesis induced by acidic and basic fibroblast growth factors (aFGF and bFGF), epidermal growth factor and VEGF, and all of these factors require angiogenin to exert their angiogenic activity. Accordingly, angiogenin crucially regulates angiogenesis by its nuclear activities in endothelial cells (Moroianu and Riordan, 1994a; Kishimoto et al., 2005; Tsuji et al., 2005; reviewed by Gao and Xu, 2008).

1.2.4 Signalling

Coordination of normal growth and development of a multicellular organism requires for cells to be able to adapt to changes in their environment and to signal to neighbouring cells. The diagram below illustrates the best studied and most common examples of signalling cascades elicited by binding of a signal molecule to its corresponding cell surface receptor (Figure 5). Besides 18 ion channels, two major types of cell surface receptors are commonly distinguished: G-protein-coupled receptors (GPCR) or receptor tyrosine kinases (RTK). G-proteins are heterotrimeric proteins that hydrolyse GTP and are therefore also referred to as GTPases. G-proteins are activated by ligand-bound GPCRs, which in their turn activate adenylyl cyclase to produce cyclic AMP (CAMP) thereby triggering protein kinase A (PKA) activity. Alternatively, G-proteins can initiate signalling via phospholipase C, an enzyme that cleaves phosphatidylinositol 4,5-bisphosphate (PI 4,5BP) to generate the second messenger molecules inositol triphosphate (IP3) and diacylglycerol. These trigger activation of calcium-dependent signalling molecules such as Ca2+/calmodulin-dependent kinase and protein kinase C (PKC). The receptor tyrosine kinases, the second major type of cell surface receptor, can similarly activate PLC or trigger kinase cascades. The two main signalling routes involve phosphatidylinositol (PI) 3 kinase, which in its turn leads to stimulation of protein kinase BlAkt (PKB) or mitogen-activated protein (MAP) kinases. Several types of MAP kinases have been identified, including the extracellular signal-regulated kinases 1 and 2 (Erkll2) or the c- Jun N-terminal kinases (JNK). Accordingly, the signalling output is highly dependent on the complex interplay of signalling cascades activated within a cell at any given time point and often involves regulation of target genes to modulate cellular activities, such as cell spreading or proliferation (summarised by Lodish, 2000; and Alberts, 2002). receator

'1 kinase

G protein G protein + phospholipase C

cyclic AMP

Figure 5: Overview of common intracellular signalling pathways in response to extracellular signalling molecules. Extracellular signalling proteins bind specific receptors on the cell surface, which can be G-protein- coupled receptors or receptor tyrosine kinases. Extracellular signals commonly lead to the activation of one or several of the signalling pathways indicated above, all leading to the activation of kinases (yellow) with context- dependent signalling outcome (Alberts, 2002). Abbreviations: CaM-kinase: Ca2+lcalmodulin-dependent kinase; GEF: Guanine nucleotide Exchange Factor; IPS: lnositol triphosphate; MAP: Mitogen-Activated Protein; PI: Phosphatidylinositol; PKA: Protein Kinase A; PKB: Protein Kinase BIAkt; PKC: Protein Kinase C

Angiogenin signalling in response to receptor binding has been shown to involve many different signalling molecules depending on the cell type investigated: In both endothelial and smooth muscle cells it has been shown to activate PLC (Bicknell and Vallee, 1988; Moore and Riordan, 1990). Furthermore, in smooth muscle cells angiogenin has been observed to stimulate SAPKIJNK MAP kinase without requiring nuclear localisation (Xu et al., 2001). Likewise, in smooth muscle cells transient depression of the CAMP 20 level was induced by angiogenin (Xiao et al., 1989). In endothelial cells Erkll2 MAP kinase stimulation was reported independently of angiogenin nuclear translocation, however in the same study no activation of SAPKIJNK MAP kinase was seen (Liu et al., 2001). Similarly, angiogenin treatment of endothelial cells triggered PKB signalling without nuclear translocation (Kim et al., 2007). The activation of PI-3 kinase and PKB in endothelial cells by angiogenin has been shown to induce the synthesis of NO, however this also depended on angiogenin nuclear translocation (Trouillon et al., 2010). Delayed stimulation of the PI-3lAkt pathway by angiogenin has also been reported in primary motoneuron cultures (Kieran et al., 2008).

Taken together, signalling activated in response to angiogenin stimulation is highly context-dependent and determined by the cell type under investigation. No further information concerning the receptor type triggering signalling can be drawn, as the 170 kDa receptor remains uncharacterised to date, and as some signalling events do not require nuclear localisation, angiogenin may be involved in other processes but angiogenesis.

1.2.5 Ribonucleolytic Activity

Ribonucleic acids (RNA) are used by cells at various steps during the production of proteins from encoding genes (Figure 6A). Initially, a gene encoded by double-stranded DNA in the nucleus is transcribed into single- stranded messenger RNA (mRNA). This mRNA molecule is cotranscriptionally processed by the spliceosome to remove any intronic sequences encoded by the transcribed gene. The core of the spliceosome itself is formed by so-called small nuclear ribonucleoproteins (snRNP), which are composed of small nuclear RNAs (snRNAs) and proteins. The mature, spliced mRNA is then transported from the nucleus to the cytoplasm for translation by the ribosome. Eurkaryotic ribosomes consist of the 40s and the 60s ribosomal subunits (Figure 6B). They are composed of one third protein and two thirds enzymatically active RNA, the ribosomal RNAs (rRNA). Functional ribosomes are assembled in the nucleolus, an organelle contained 2 1 within the nucleus not bound by a lipid membrane. The sequence encoded by the mRNA is translated into a protein with the aid of aminoacyl transfer RNA (tRNA). These tRNA molecules bind ribosomes and are attached to the corresponding amino acid encoded by the complementary mRNA codon, which is recognised by the anticodon af the tRNA (Figure 6C). Ribosomes catalyse the formation of peptide bonds between amino acids encoded by the mRNA molecule (summarised by Lodish, 2000; and Alberts, 2002). Figure 6: Different types of RNA molecules contribute to the process of protein synthesis from the encoding gene. A) Schematic representation of the steps involved in protein synthesis starting with DNA transcription into messenger RNA (mRNA) via processing of the primary transcript by splicing of intronic sequences. Mature mRNA is exported from the nucleus and translated into a protein with the aid of ribosomes (Alberts, 2002). The ribosomes consist of one large and one small subunit (B), which are both formed from ribosomal RNA (rRNA; orange and yellow) and protein (blue; www.pdb.org). Ribosomes bind mRNA and the coded sequence is recognised by transfer RNA (tRNA, C) molecules attached to the corresponding amino acid (yellow), also referred to as aminoacyl tRNA (Alberts, 2002). Angiogenin displays four to six times lower RNase activity than RNase A, but its RNase activity is essential for its biological function of angiogenesis induction (Shapiro et al., 1986; Shapiro et al., 1987a).

Experiments performed in vitro have shown that angiogenin cleaves rRNAs leading to inhibition of protein synthesis in a cell-free system (St Clair et al., 1987). Despite the blocked substrate binding site of angiogenin detailed above, it displays higher ribonucleolytic activity on rRNAs in ribosomal particles than other RNases (Shapiro et al., 1986). The base specificity of RNA cleavage by angiogenin was investigated in comparison with RNase A and it was reported that each enzyme displays a unique cleavage pattern of natural RNAs (Rybak and Vallee, 1988). Angiogenin activity on specifically the 40s subunit of the ribosome was observed to be responsible for the inhibition of protein synthesis (St Clair et al., 1988).

The RNase activity was further studied using in vitro assays on a variety of RNA substrates, including tRNAs (Shapiro et al., 1987a; Lee and Vallee, 1989). Injection of RNases into Xenopus oocytes abolishes protein synthesis without generating rRNA cleavage products, which led the authors to speculate on the existence of a more specific RNA target (Saxena et al., 1991). Later, it was shown that angiogenin causes degradation of tRNAs with toxic effects on cells (Saxena et al., 1992). Recent reports show that angiogenin is required for the production of stress-induced small RNAs (tiRNA) from tRNAs resulting in translational repression independent of the classical phospho-elF2a-dependent stress response programme (Fu et al., 2009; Yamasaki et al., 2009). The observed tRNA fragments appear in a variety of mammalian cell types in response to stresses such as heat shock, hypothermia, hypoxia or irradiation and correspond to tRNA halves produced from fully mature tRNAs by cleavage within the anticodon loop (Fu et al., 2009). The second study on stress-induced tRNA cleavage by angiogenin observed that angiogenin activity was normally repressed by its inhibitor RNHI and established a direct link between the 5' tiRNA fragments generated and translational repression. Interestingly, stress-induced tRNA cleavage was independent of nuclear angiogenin translocation (Yamasaki et al., 2009). Very recently, the mechanism of inhibition of protein translation by tiRNAs was shown to involve binding and displacement of the initiation factors elF4G and elF4A from mRNAs (Ivanov et al., 201 1).

Angiogenin's role in stress responses therefore gives one explanation for its non-angiogenic activities that are independent of nuclear localisation but have long been suggested based on the protein's widespread expression (Moenner et al., 1994).

1.2.6 Angiogenin in Angiogenesis

Angiogenesis is a complex process, which requires for endothelial cells present in intact blood vessels to migrate across the basement membrane surrounding the vessel into the neighbouring tissue. Endothelial cell migration is guided by stimuli such as angiogenin and aided by proteases modifying the extracellular matrix (ECM).

Sequencing of many RNase proteins from various species confirmed sequence identity with angiogenin between 30 to 70 % and some of them have been tested for angiogenic activity. Interestingly, angiogenin remains the only RNase able to induce angiogenesis, which has been attributed to a missing fourth disulfide bond when comparing the structure of angiogenin to that of other RNases (reviewed by Adams and Subramanian, 1999; and Tello-Montoliu et al., 2006). Angiogenin may contribute to this process by multiple mechanisms: Angiogenin can stimulate endothelial cell proliferation, migration and tubule formation (Jimi et al., 1995; Hu et al., 1997), it supports adhesion of endothelial cells and fibroblasts on coated plastic (Soncin, 1992) and may contribute to the ECM (reviewed by Strydom, 1998). It was observed that HT-29 cells adhere to angiogenin in a heparin- and heparinase-sensitive way, indicating the contribution of proteoglycans to cell adhesion (Soncin et al., 1994). Angiogenin binding to actin at the cell surface (Hu et al., 1991; Hu et al., 1993), causes dissociation of the angiogenin-actin complex from the plasma membrane and activation of tissue-type plasminogen activator, which in turn converts plasminogen to plasmin (Hu and Riordan, 1993). Plasmin activates collagenases required for angiogenesis (reviewed by Strydom, 1998; and Gao and Xu, 2008). A complex regulatory network has been suggested: Plasminogen is a substrate of elastase leading to the generation of angiostatin, an inhibitor of angiogenesis. Aided by plasminogen, elastase also cleaves angiogenin to a product that is still ribonucleolytically active but has lost its ability to be internalised into the nucleus of endothelial cells. Nevertheless, the meaning of this regulative circuitry remains to be firmly established (Hu, 1997; reviewed by Strydom, 1998; and Gao and Xu, 2008).

In conclusion, angiogenesis may be regulated by angiogenin at many different steps: It binds endothelial cells, is internalised by them and triggers signalling pathway activation. Endothelial cells proliferate and form tubular structures in response to angiogenin signalling. Various cell types have been shown to adhere to angiogenin and extracellular proteases are activated by it, thereby initiating cell invasion (reviewed by Strydom, 1998; and Gao and Xu, 2008).

1.2.7 Angiogenin Mutations in ALS

Following the observation that deleting the hypoxia-reponse element in the VEGF promoter causes degeneration of motoneurons reminiscent of ALS (Oosthuyse et al., 2001) and the identification of single nucleotide polymorphisms in the VEGF promoterlleader sequence associated with increased risk of developing ALS in Swedish, Belgian and English individuals (Lambrechts et al., 2003), it was speculated that other genes involved in regulating cellular responses to hypoxia could be mutated in ALS patients. Recently, the angiogenin gene was found to be mutated in Irish and Scottish patients with both familial and sporadic ALS in a study performed on Irish, British, American and Swedish patients (Greenway et al., 2004; Greenway et al., 2006). The study identified seven missense mutations (Figure 7), five of which involving functionally important residues with regard to angiogenin's ribonucleolytic activity in regions highly conserved between RNase A family 26 members. One mutation was speculated to affect nucleolar targeting, while the last mutation was predicted not affect the protein's structure majorly (Greenway et al., 2006).

Figure 7: Representation of the three dimensional structure of angiogenin with the original mutations found. The missense mutations (yellow and turquoise) Q12L, K171, K17E, C39W and K401 are contained within highly conserved regions of the protein thus affecting functionally important residues for angiogenin's ribonucleolytic activity. The R31K mutation is located in the nucleolar localisation signal, while the 146V mutation does not affect the proteins structure majorly (Greenway et al., 2006).

This initiated the search for further mutations in the angiogenin gene in populations of different origin: Three additional loss-of-function mutations were identified in North American ALS patients (Wu et al., 2007). Another six novel mutations in the angiogenin gene were characterised studying Italian ALS patients (Conforti et al., 2008; Gellera et al., 2008). However, two other studies on Italian ALS patients did not report mutations in the angiogenin gene to be associated with ALS (Corrado et al., 2007; Del Bo et al., 2008). Three further novel angiogenin mutations were identified in studies on French and German ALS patients (Paubel et al., 2008; Fernandez-Santiago et al., 2009). Nevertheless, no association of angiogenin mutations with ALS pathology was established in Polish patients (McLaughlin et al., 2010).

The identified mutations affect angiogenin's ribonucleolytic activity, its nuclear localisation or have been located to its signal peptide sequence. For some mutations functional changes have been characterised (Crabtree et al., 2007; Wu et al., 2007), while for few of them the pathological mechanism remains elusive or based on modelling predictions. One mutation (146V) has

27 been identified in several screens (Greenway et al., 2006; Gellera et al., 2008; Paubel et al., 2008; Fernandez-Santiago et al., 2009), but also in healthy Italian controls (Corrado et al., 2007; Conforti et al., 2008), so it has been speculated that it could be a benign mutation.

1.2.8 Angiogenin in ALS Pathomechanism

Since the discovery of angiogenin mutations in ALS patients, studies have been performed to identify the protein's role in ALS pathology. Angiogenin protein can be purified from normal human blood and bovine milk (Shapiro et al., 1987b; Maes et al., 1988). Angiogenin circulates in human plasma at a concentration range between 250 and 360 ng/mL (Blaser et al., 1993; Shimoyama et al., 1996) and has been observed to increase in different pathological conditions, particularly in cancer (reviewed by Tello-Montoliu et al., 2006).

Several studies have investigated angiogenin levels in the plasma of ALS patients: Elevated serum angiogenin but normal VEGF levels were observed in a study performed on Irish patients (Cronin et al., 2006). Later however, a larger study by the same group found serum angiogenin levels to be significantly lower in ALS patients (McLaughlin et al., 2010). Interestingly, serum angiogenin levels normally allow for prediction of angiogenin levels in the cerebrospinal fluid (CSF), a correlation shown to be lost in ALS patients in this study, indicating a tissue-specific mechanism of regulation of angiogenin levels. Another study on angiogenin levels in CSF of ALS patients did not report changes compared to healthy controls (Ilzecka, 2008). Investigating angiogenin levels in CSF of French ALS patients showed that patients fail to upregulate angiogenin with hypoxaemia, pointing to defective responses to hypoxia mediated by hypoxia-inducible factor la(Moreau et al., 2009). Taken together, regulation of angiogenin levels in plasma and CSF of ALS patients may be altered by complex mechanisms depending on many factors such as disease stage and severity. 1.2.9 Angiogenin Effects on Motoneurons

In spinal cord ventral horn motoneurons from both normal adult and fetal human autopsies strong staining for angiogenin protein was detected (Wu et al., 2007). As angiogenin is a secreted protein, the same study also detected strong angiogenin staining in the ECM and the interstitial tissue. Furthermore, angiogenin was found to be present in endothelial cells lining blood vessels of the spinal cord. Angiogenin expression in mouse spinal cord motoneurons was confirmed both in vivo (Greenway et al., 2006; Kieran et al., 2008) and in vitro (Greenway et al., 2006).

Several studies have been performed attempting to elucidate the mechanism of action of angiogenin in motoneurons: It has been observed that administration of recombinant human angiogenin protein to SODl mutant mice increased life span and motoneuron survival, which is correlated with better motor function (Kieran et al., 2008). SODl mutant mice display decreased levels of the active kinases Akt-I and Erkll2 and increased expression of intercellular adhesion molecule-I, a marker for activated endothelium. All three effects were reversed by angiogenin administration. Investigation of the neuroprotective properties of angiogenin in vitro by the same study showed that it protects motoneurons from excitotoxic and ER stress-induced cell death, an effect attributed to activation of PI-3 kinase. Similarly, angiogenin protected primary mouse motoneurons against hypoxic injury (Sebastia et al., 2009). Under hypoxic conditions both wildtype and SODl mutant motoneurons upregulated angiogenin in a hypoxia-inducible factor-I a-dependent manner. However, the ALS-associated mutants of angiogenin had lost their protective effects in a motoneuron-like cell line.

During embryonic CNS development high expression levels of murine angiogenin-I were detected predominantly in neurons (Subramanian and Feng, 2007). Angiogenin expression decreased during development but remained detectable in adulthood. The authors investigated angiogenin effects on neuronal differentiation in a cell culture model and observed that committed neuroectodermal precursors started expressing angiogenin, which accumulated in neurites and cell bodies of neurons. lnhibiton of angiogenin function inhibited neurite pathfinding but not neuronal differentiation. Three of the ALS-associated angiogenin variants with reduced ribonucleolytic activity were later observed to affect neuronal pathfinding and had cytotoxic effects on motoneurons (Subramanian et al., 2008). Additionally, the angiogenin mutants investigated had lost their ability to confer neuroprotection in cell culture. Furthermore, neuroprotective effects of angiogenin on two neuroblastoma cell lines under oxidative stress were observed and angiogenin stimulated cell migration (Cho et al., 2010). The ALS-associated angiogenin mutant with lowest ribonuclease activity tested, did not protect the cell lines from oxidative stress and was unable to stimulate migration. Angiogenin was similarly observed to exert anti-apoptotic effects in an embryonal carcinoma cell line subjected to serum withdrawal, another commonly used cell stress paradigm (Li et al., 2010). The authors further attribute the anti-apoptotic effects described to angiogenin upregulating the anti-apoptotic protein Bcl-2 leading to blocked cytochrome c release from mitochondria and inhibition of caspase activation, both crucial steps in the execution of apoptosis. The most recent study on the same embryonal carcinoma cell line found that angiogenin inhibited cleavage of the apoptosis-inducing factor poly ADP- ribose polymerase-I and thereby prevented its nuclear translocation from mitochondria by upregulating Bcl-2, which prevents caspase-3 activation (Li et al., 201 1).

In conclusion, angiogenin's role in ALS pathology remains elusive, but many studies have investigated its neuroprotective properties both in vitro and in vivo, making angiogenin an interesting target for ALS therapy. 1.3 Aims of the Study

Presently, the role of angiogenin in ALS pathology and normal brain physiology is not very well established. This study was performed to further investigate the interaction between primary astrocytes and motoneurons in cell culture. Particular focus of this study was put on the role of angiogenin with regard to intercellular communication.

The aims of the study were:

To elucidate the uptake mechanism of angiogenin into the different cell types in primary mouse cell cultures derived from the central nervous system

To characterise the pathway of vesicular angiogenin internalisation by astrocytes in primary cultures

To identify the angiogenin receptor on primary mouse astrocytes and the signalling response following receptor binding

To investigate the secretory profile of primary mouse astrocytes by mass spectrometry

To determine the effects of angiogenin on the astrocyte secretome in response to angiogenin treatment

To validate mass spectrometric results focusing on neuroprotective candidate proteins 2. Materials and Methods

2.1 Materials

2.1 .IReagents

Chemical Catalogue no Manufacturer

Acetic acid 201 04.298 VWR Acetonitrile 1.00030.1 000 Merck Acrylarnide 40 % 17-1303-01 PlusOne Acrylamidelbis-acrylamide 40 % A71 68 Sigma Agarose 5008 1 Lonza Ammonium bicarbonate A6141 Sigma Angiogenin 265-AN R&D Systems APS 17-131 1-01 PlusOne B27 supplement 17054 Gibco P-glycerophosphate G6251 Sigma Bis-acrylamide 2 % 17-1306-01 PlusOne Braun water Aqua B. Braun B Braun Brornophenol Blue B8026 Sigma BSA A21 53 Sigma CaCI2 C1016 Sigma Cacodylate sodium 214970250 Acros Organics Ciliary neurotrophic factor 557-NT R&D Systems Colchicine C9754 Sigma Complete protease inhibitors 11873580001 Roche D-glucose G8769 Sigma-Aldrich Dialysed serum 14-810F Lonza Diethylether 2381 1.292 VWR DMEM 12-604F Lonza DMEM -Arg/Lys 89985 Pierce Dnase l DN-25 Sigma DTT EU0006-D Eurornedex Dynasore D7693 Sigma ECL Plus Western blotting detection reagents RPN2132 GE Healthcare EDTA El644 Sigma EGTA E4375 Sigma Ethanol 20820.327 VWR Fetal bovine serum F7524 Sigma Formic acid 1.00264.1000 Merck Fungizone 15290-018 lnvitrogen Glial cell line-derived neurotrophic factor G2781 Promega Glutaraldehyde 16220 Electron Miroscopy Sciences Glycerol PlusOne Glycine Biomol Heavy ArglO (L-[U-13C6-15N41-Arginine) Cambridge Isotope Heavy Lys8 (L-[I 3C6-15N21-Lysine) Laboratories Heparin Sigma Heparinase I Sigma Horse serum Gibco lmmobilon Western Chemiluminescent HRP Millipore Substrate lsopropanol 20842.298 VWR KOH 26669.266 VWR L15 medium 1141 5-049 lnvitrogen Laminin L2020 Sigma Lead nitrate 26 554.293 WVR L-Glutamine G7513 Sigma Lipofectamine 2000 11668-01 9 lnvitrogen LysoTracker Red L7518 Biosciences Medium Arg6 (L-[U-13C61-Arginine) CLM-2265 Cambridge Isotope Medium Lys4 L-[2H4]-Lysine DLM-2640 Laboratories Methyl-p-cyclodextrin C4555 Sigma Micro BCA Protein Assay Kit 23235 Pierce Milk powder dried skimmed milk Minimum essential medium M4526 Gibco Monodansylcadaverine 30432 Sigma NaCl 71381 Fluka NaCl S9888 Sigma NaF S6776 Sigma NaOH 28145.265 VWR Neurobasal 21103 Sigma NP-40-Tergitol8 NP40S Sigma NuPAGE MOPS SDS Running Buffer 20 X NP0001-02 lnvitrogen Optimem 31985-047 Gibco Osmium tetraoxide 19150 Electron Microscopy Sciences Paclitaxel Sigma PAGE Blue Fermentas PAGE Ruler Plus Fermentas Paraformaldehyde Riedel-de Haen Paraformaldehyde (16 %) Electron Microscopy Sciences PBS tablets Sigma PenicillinlStreptomycin Sigma Pentobarbitone DoLethal Vetoquinol Phalloidin FlTC Sigma Phosphatase inhibitor cocktail 1 Sigma Phosphatase inhibitor cocktail 2 Sigma Poly-L-Ornithine Sigma Ponceau Sigma Prestained protein ladder Euromedex Progesterone Sigma Protease inhibitors Sigma Protein AIG PLUS-Agarose Santa Cruz SDS Sigma SDS 10% BioRad Sodium citrate Electron Microscopy Sciences Sodium orthovanadate Sigma Sodium pyrophosphate Sigma Sucrose Sigma TEMED Sigma Trichloroacetic acid Prolabo Tris PlusOne Triton XI00 Fluka Triton XI00 PlusOne Trizma Sigma Trypsin Sigma TrypsinIEDTA Sigma Trypsin Gold Promega Tween 20 Sigma Uranyl acetate Prolabo Vectashield Vector Labs

2.1.2 Equipment

Consumable Manufacturer Model

4-1 2 % Bis- lnvitrogen NP0321 BOX PTrispolyacrylamide gel 6-well plates Greine Bio-One 8-well chamber slides IBlDl 24-well plates Sarstedt Ltd. 83.1836 Copper grids Gilder Grids GI00HEX-C3; 0 3.05mm; hexagonal; 100 lineslinch Coverslips 0 13 mm VWR International 631-0149 Duolink II Red starter kit Olink 92101 Endofree Plasmid Purification Qiagen 12362 Maxi Kit Epon IM Resin Electron Microscopy Sciences Eppendorf tubes for MS Trefflab 1.5 mL Click Fit Polyproylene 96.0751 4.9.01 HPLC precolumn Pepmap@ 0.3 mmx 10 mm HPLC reverse-phase column Pepmap@ 0.075 mm x 150 mm Nitrocellulose Fisher Scientific BRD-100-520C Nitrocellulose Amersham Biosciences Hybond-C Extra RPN 303E Photographic film Kodak BioMax Light Film; Scientific imaging film Polysine microscope slides VWR International 631-0107 Scissors World Precision Instruments Vannas Scissors; straight; 5 mm blades Superfrost microscope slides Thermo Menzel LR90SF02 Surgical scalpel blades Swann-Morton Caron steel 0201 T75 tissue culture flask BD Falcon 353136 Tweezers: curved World Precision Instruments Dumont Tweezers #7 curved; Dumoxel straight Dumont Tweezers #5 straight; Dumoxel Whatman chromatography Whatman 3MM CHR paper

Instrument Manufacturer Model

Big gel electrophoresis BioRad Protean II xi Cell system Chemiluminescent imager FujiFilm LAS 3000 34 Confocal microscope Zeiss LSM 710 Confocal microscope Zeiss LSM 780 Dissection microscope Olympus SZ5 1 Electrophoresis system BioRad Mini-Protean Tetra Electrophoresis system lnvitrogen XCell Sure Lock E100001 Fluorescent microscope Nikon TE 2000-S Fluorescent microscope Zeiss Axiolmager HPLC Thermo Fisher Scientific U3000 nanoflow Laminar flow hood ESI Flufrance Cytair 128 Mass Spectrometer Thermo Scientific LTQ Orbitrap XL Mass Spectrometer Thermo Scientific LTQ Orbitrap Velos Osmometer Knauer K-7400 Semimicro Osmometer Plate Reader Tecan GENios Scanner Epson V750 PRO Semi-Dry Transfer Cell BioRad TransBlot SD Spectrophotometer Thermo Scientific NanoDrop 2000 Thermomixer Eppendort 1.5 mL Transmission electron JEOL Ltd 1200 EX1 l microscope Ultramicrotomy Leica Ultracut UCT with Stereo Zoom 6 Photo 2.2 Methods

2.2.1 Cell Culture

2.2.1 .I Preparation of Primary Motoneuron Cultures

All primary motoneuron cultures prepared for immunocytochemistry were seeded onto precoated coverslips in 24-well plates to aid motoneuron adhesion on a glass surface. Poly-L-Ornithine was prepared in sterile phosphate buffered saline (PBS) at a final concentration of 1.5 pglmL and 400 pL were added per well over night at room temperature (RT). The solution was aspirated and the wells washed once with sterile PBS. Laminin was prepared in L15 medium at a concentration of 2.5 pg/mL and 400 pL were added per well for 2 h at 37 "C. The medium was aspirated, the wells washed once again with sterile PBS and the plate stored at 4 "C until cell seeding.

To ensure optimal preparation and growth conditions for motoneurons, two different types of medium were prepared freshly before every motoneuron preparation: complete medium for tissue dissociation and full Neurobasal (NB) for optimal growth in culture.

Table 1: Primary motoneuron media Complete medium Full Neurobasal

49.4 mL Neurobasal 47.5 mL Neurobasal 500 pL PenicillinIStreptomycin (PIS) 1 mL Horse serum 125 pL L-Glutamine (Q) 1 mL B27 500 pL PenicillinIStreptomycin 125 pL L-Glutamine 10 pL Ciliary neurotrophic factor (5 pg1mL) 10 pL Glial cell line-derived neurotrophic factor (10 ~g/mL) 50 pL Fungizone Primary motoneuron cultures were prepared from El3 C57 Black 6 mouse embryos (Camu and Henderson, 1992). Pregnant mice were intraperitoneally given a lethal dose of pentobarbitone (40 mglkg). Sedation of the mouse was assessed by pinch-reflex. To remove the embryos, hysterectomies of the uterus were carried out. The embryos were kept on ice in sterile PBS during dissection. After decapitating the embryos, the ventral horns were dissected using a dissection microscope. The embryo was held face forward with curved tweezers and the skin covering the developing, unclosed spinal cord was carefully removed with a second pair of tweezers. The roots of the nerves emanating from the spinal cord to the body were carefully snipped before removing the two body halves one after the other. The meninges were carefully removed from the ventral face of the spinal cord. By holding the spinal cord carefully at the brain stem, the dorsal horns were removed from the oral to the caudal end.

After dissecting the ventral horns, excess PBS was removed from the tissue and it was digested in I mL 0.025 % trypsin in PBS for 10 min at 37 "C. The trypsin solution was carefully aspirated and the tissue transferred into a new tube containing 800 pL complete medium. After adding 100 pL 4 % (wlv) bovine serum albumin (BSA in complete medium; 0.4 % final) and 100 pL DNase I (I mg/mL in complete medium; 0.1 mglmL final), cells were carefully dissociated by trituration. The cell solution was left to settle for 2 min before collecting the supernatant into a new tube. Trituration was repeated twice in 1 mL complete medium containing 0.4 % BSA and 0.02 mglmL DNase I. To protect motoneurons during centrifugation, 1 mL of 4 % BSA was added to the collected supernatant containing dissociated cells before spinning at 1500 rpm for 5 min. The supernatant was discarded, the cells counted and 250,000 viable cells in 500 pL full Neurobasal were seeded per well of a 24-well plate containing precoated coverslips. Cells were cultured at 37 "C with 5 % C02supply.

As motoneurons only adhere very weakly to the coverslip, the medium was very carefully replaced by removing 200 pL and adding 300 pL of fresh full Neurobasal to ensure sufficient nutrient supply for motoneurons. After 7 days, cultures were treated with recombinant human angiogenin protein (1 pg1mL) for I h and prepared for immunocytochemistry as described below (pg. 42).

2.2.1.2 Preparation of Primary Astrocyte Cultures

To ensure optimal growth of primary astrocytes, the cells were cultured in full Dulbecco's Modified Eagle Medium (DMEM; 4.5 g/L glucose) containing 2 mM L-Glutamine, 100 UlmL PIS and 10 % fetal bovine serum (FBS).

Primary astrocyte cultures were prepared from postnatal day 2 (P2) C57 Black 6 mouse pups. First, pups were decapitated, the skin carefully cut along the midline of the skull starting at the caudal end to the eyes and removed to reveal the skull. The soft bone covering the brain was cut starting rostrally between the eyes along both sides of the head to obtain access to the brain. The olfactory bulb and the brain stem were carefully detached using a pair of curved forceps and the brain scooped into sterile PBS on ice for storage during dissection. With the help of a dissection microscope, cortices were dissected by cutting the brain into two halves and removing the thalamus, before carefully taking off the meninges covering both cortices using a pair of straight forceps. The cortices were cut into 6 to 8 pieces each and excess PBS was removed from the tube.

The tissue was digested in Minimum Essential Medium (6 mL for 5 or 6 pups) containing 0.025 % trypsin and 0.1 mglmL DNase I for 15 min at 37 "C. The DNase was added to digest the DNA of destroyed cells, which otherwise forms long strings interfering during trituration of the tissue. Before centrifuging at 1000 rpm for 5 min, the same volume of full DMEM was added to the tube to buffer the activity of the trypsin. The supernatant was discarded and the tissue was carefully triturated three times in 5 mL full DMEM containing 0.1 mglmL DNase I to dissociate individual cells. Astrocytes were plated in 10 mL full DMEM per T75 tissue culture flask (2 flasks for 5 or 6 pups) and cultured at 37 "C with 5 % C02supply. After 1 wk in culture, the cells were washed once with sterile PBS and 10 mL fresh full DMEM were added per T75 flask. Full confluence of the flask was usually reached after 2 wk in culture, which allowed for one passage of the culture for plating in the appropriate assay format. For passaging, the cells were washed once in sterile PBS before adding 4 mL trypsinlEDTA. The cells were incubated at 37 "C for 5 min to detach the cells from the flask, collected into a new tube by adding 6 mL full DMEM to quench the trypsin and the flask was rinsed once with 5 mL full DMEM to ensure complete cell collection. After spinning the cells down at 1000 rpm for 5 min, the supernatant was discarded, the cells resuspended in full DMEM (10 mL per flask) and counted. For immunocytochemistry, the cells were seeded onto poly-L-Ornithine-coated coverslips in 24-well plates at 250,000 cells per well in 500 pL full DMEM. Western blotting was performed on cells seeded at 1 Mio cells per well of a 6-well plate in 2 mL full DMEM or 500,000 cells per well of a 12-well plate in I mL full DMEM.

2.2.1.3 NSC34 Cells

NSC34 cells, a cell line with primary motoneuron-like characteristics, is a hybrid cell line derived from mouse neuroblastoma cells and embryonic mouse spinal cord (Cashman et al., 1992). These cells were routinely kept in full DMEM in T75 flasks as described for primary astrocyte cultures. They were passaged as described above at about 80 % confluence, as their viability rapidly deteriorated at greater confluence. Cells were reseeded at 1 in 5 or 1 in 6 in T75 flasks depending on the viability and density before passaging.

NSC34 cells were seeded onto poly-L-Ornithine-coated coverlips in 24-well plates after passaging at 1 in 4 and 1 in 8, the more viable cells were then chosen for transfection and immunostaining. The cells were seeded at two densities, as they do not adhere well to the coverslips, so they should be at optimal density of approximately 60 % when transfecting. 2.2.2 Angiogenin Uptake Studies

Angiogenin treatment of primary cell cultures to determine angiogenin uptake by immunocytochemistry was routinely performed on astrocytes two days after seeding into 24-well plates containing poly-L-Ornithine-coated coverslips. Cells were washed three times with sterile PBS before adding 500 pL Neurobasal medium (containing only PIS and Q) per well and 5 pL of recombinant human Angiogenin (rhAng) diluted in sterile PBS at a concentration of 0.1 mglmL for 1 h. For Western blotting the volumes were adjusted depending on the plate format. The final concentration of I pglmL and the timing was chosen to ensure good visualisation of angiogenin uptake by immunocytochemistry.

2.2.2.1 Organelle Markers to Monitor lntracellular Trafficking

Two organelle markers were used in this study to follow the route of endocytosed rhAng. LysoTracker Red (LTR) accumulates specifically in acidic organelles, mainly lysosomes, as it consists of a fluorophore attached to a weak base that can permeate cell membranes and upon protonation remains within the organelles. It can be used for both, labelling of cells prior to fixation or POP live cell imaging. Monodansylcadaverine (MDC) is an autofluorescent compound similarly labelling acidic vesicles like lysosomes (Biederbick et al., 1999; Niemann et al., 2000) as well as autophagic vacuoles (Biederbick et al., 1995).

Astrocytes were treated with rhAng as described above. Treated cells were labelled with 50 nM LTR for 30 min. Similarly, 50 pM MDC was added 10 min prior to fixation. Cells were fixed and colocalisation analysis of angiogenin containing vesicles with other vesicular markers was performed using a LSM 710 confocal microscope with Zen 2009 software. 2.2.2.2 Inhibition of Endocytosis

To study the endocytic mechanism utilised by astrocytes to take up rhAng, astrocyte cultures were pretreated with several different compounds. Cell cultures were washed three times in sterile PBS before adding 500 pL of fresh Neurobasal (containing PIS and Q) with the following inhibitors for the indicated pretreatment times: Angiogenin endocytosis was manipulated by adjusting the salt concentration and osmolarity of Neurobasal medium to DMEM levels using NaCl (3 glL to 6.4 g/L), CaCI2 (200 mg1L to 264 mg1L) and sucrose (235 mOsm to 335 mOsm) and pretreating the astrocytes for 3 h. Sucrose was added assuming that a 1 mM change in sucrose concentration in the medium changes the osmolarity by 1 mOsm. The actual osmolarity was confirmed using an osmometer and recorded for every experiment. The modified medium was sterile filtered prior to cell treatment. To elucidate the mechanism of angiogenin uptake, endocytosis was manipulated by pretreating astrocytes for I h with 0.2 mM monodansylcadaverine (MDC) or 0.24% methyl-P-cyclodextrin (MPCD; wlv) to distinguish clathrin- and lipid raft-dependent endocytic events, respectively. Pretreatment of astrocytes with dynasore for 30 min at both 40 and 160 pM was performed to test for the involvement of the G-protein dynamin and to rule out cytotoxic effects of dynasore treatment. The contribution of heparansulfate proteoglycans to the endocytic mechanism was tested by pretreating astrocytes for 2 h with heparinase I at 2.5 UlmL or heparin at 10 pg/mL. To test whether angiogenin uptake by astrocytes required an intact microtubule network, astrocytes were pretreated for I h with the microtubule stabilising paclitaxel at Ipg/mL or the microtubule disrupting colchicine at 4 pglmL. The uptake of 1 pg1mL angiogenin was determined by adding the recombinant protein after preincubation for 1 h before cell fixation.

Analysis of immunocytochemistry was carried out by preparing two coverslips of each treatment condition stained using antibodies against the astroglial marker GFAP and angiogenin, taking at least three random images per coverslip at 60X magnification using either an epifluorescent or pseudoconfocal microscope as indicated. The number of cells containing angiogenin vesicles and those staining positive for GFAP was counted manually with the help of the AlphaEaseFC 4.0 software. Counts were tested for statistical significance between three separate experiments using Student's t-test.

2.2.3 lmmunocytochemistry

lmmunocytochemistry is a technique commonly used to study protein localisation and trafficking, as well as to quantify protein expression. Primary antibodies specifically targeting a protein of interest within fixed and permeabilised cells first bind the protein. This interaction can then be detected by using a secondary antibody, raised against the species of the primary antibody and conjugated to a fluorescent label such as rhodamine, FlTC or Alexa dyes. By using primary antibodies from different species and appropriate, secondary antibodies with distinct fluorescence spectra, multiple proteins can be stained and colocalisation can be analysed.

2.2.3.1 lmmunostaining lmmunocytochemistry was performed on astrocyte and motoneuron cultures seeded on coated coverslips in 24-well plates. Cells were carefully washed three times in ice cold PBS and fixed for 20 min in 400 pL 4 % paraformaldehyde (PFA; wlv) in PBS at RT. After three washes in PBS, the cells were blocked in 5 % milk (wlv) in PBSlO.1 % Triton XI00 for 1 h at RT to reduce non-specific antibody binding. Cultures were stained with primary antibodies (Table 2) diluted in blocking solution over night at 4 "C, followed by two washes in PBSlTriton and one wash in PBS. All following steps were performed while protecting the samples and reagents from light: Cells were stained with secondary antibodies (Table 3) diluted I in 500 in blocking solution for 1 h at RT. After washing twice in PBSITriton and twice in PBS, coverslips were dipped into deionised water (dH20) and mounted in Vectashield containing 4',6 diamidino-2-phenylindole (DAPI) to visualise the cell nuclei. 42 Table 2: Primary antibodies for immunocytochemistry Antigen Catalogue no Origin Manufacturer Dilution

Anti angiogenin Goat Merck Anti angiogenin Mouse Abcarn Anti CDl Ib Rat Abcarn Anti clathrin HC Rabbit Abcarn Anti FAK Rabbit Santa Cruz Anti flotillin 1 Rabbit Abcarn Anti GFAP Rabbit Sigma Anti NeuN Mouse Chemicon Anti PKC Mouse Santa Cruz Anti rab 5 Rabbit Abcarn Anti rab 9 Mouse Abcarn Anti rab 11A Mouse Abcarn Anti syndecan 4 Rabbit Abcarn Anti tubulin Mouse Sigma

Table 3: Secondary antibodies for immunocytochemistry Anti-serum Catalogue no Origin Conjugate Manufacturer Dilution

Anti goat 705-295-1 47 Donkey rhodamine Jackson IR I1500 Laboratories Anti rabbit 71 1-295-152 Donkey rhodamine Jackson IR 11500 Laboratories Anti rabbit 71 1-095-152 Donkey FlTC Jackson IR 11500 Laboratories Anti mouse 715-095-1 50 Donkey FlTC Jackson lR 11500 Laboratories

2.2.3.2 Fluorescence Microscopy to Visualise lmmunocytochemistry

lmmunocytochemical staining requires for fluorescence microscopy to analyse the experimental result. The fluorescent labels used are excited by light of a specific wavelength and upon excitation emit light of longer wavelength and therefore lower energy, as the excited electrons return from high energy levels back to their ground state. Commonly, the sample is excited by using a Mercury or Xenon lamp emitting light between 300 to 1000 nm. The excitation light is focused onto the sample via a dichroic mirror, which usually reflects light of short wavelength while letting longer wavelength light pass. This results in the emission of light of longer wavelength from the sample. The light passes through an emission filter of specific wavelength to remove interfering light. The emitted and filtered light is then detected by a charge coupled device, which converts the photons into an electrical signal. In this study, fluorescence microscopy was used to elucidate the mechanism of uptake of angiogenin into astrocytes and to follow the protein's route through the cell by various colabelling strategies.

For colocalisation analysis both pseudoconfocal and confocal microscopy were used to obtain images from only one plane within the cell. In epifluorescent microscopy, the image is often blurred by signal originating from regions that are not in the focal plane. Blurring can be reduced by structured illumination by inserting a grid into the illumination pathway. The specimen is then imaged three times with slightly different grid positions. Out of focus image information from the non-illuminated dark stripes is removed by a microscope-specific software resulting in one optical section. A more advanced method to collect light from only one focal plane of the sample is confocal microscopy. This requires for the use of high power lasers to excite the fluorophores, which increases the signal from the plane in focus. The emitted light is passed through emission filters and through the confocal aperture or pinhole. Only light of specific wavelength from one plane of the sample passes the pinhole resulting in sharply focused images.

Especially, when using high power lasers or long exposure times, fluorescent samples bleach, because they are chemically damaged by photons and permanently lose the ability to fluoresce. It is therefore essential to optimise signal intensity and laser power or exposure time. To limit the effects of bleaching in this study, for all quantification experiments laser power or exposure time was kept constant throughout the experiment and the order in which the different channels were imaged was not changed. 2.2.4 Electron Microscopy

In the 1930's the first electron microscopes were built with the first commercial transmission electron microscope produced by Siemens in 1939. Electron microscopes have greater resolving power than light microscopes, as their resolution is not limited by the wavelength of photons, and can therefore achieve better than 50 pm resolution (Erni et al., 2009). Electron microscopes focus an electron beam onto the sample, which needs to be very thin sections of specimens. This requires for the samples to be fixed, dehydrated and embedded into a polymer resin for sufficient stabilisation. Image contrast is generated by staining the sections with heavy atom labels such as lead or uranium, as they scatter electrons more efficiently.

2.2.4.1 Cell Fixation and Embedding

In this study, like for immunocytochemical protein detection, astrocytes were seeded in 24-well plates onto poly-L-Ornithine-coated coverslips at a seeding density of 250,000 cells per well and treated with 1 pg1mL rhAng for 1 h in Neurobasal (with PIS and Q). For fixation, 400 pL of 2 % glutaraldehyde in 0.1 m cacodylate sodium (pH 7.4) were added after aspirating the medium from the wells without any washes to avoid disrupting cell structure. After 2 h of fixation at RT, the cells were washed in 500 pL 0.1 M cacodylate sodium at 4 "C over night. Postfixation was carried out by carefully adding 400 pL of 1 % osmium tetraoxide in 0.1 M cacodylate buffer per well straight after removing the wash buffer well by well. Following 90 min of postfixation at RT, the cells were carefully dehydrated by sequentially increasing the ethanol (EtOH) content of the washes before adding EponTMresin at RT: Table 4: Dehydration for electron microscopy Solution Incubation time No of repeats 50 % EtOH 5 rnin 2 70 % EtOH 5 min 2 96 % EtOH 10 min 2 100 % EtOH 15 min 3 50 % EtOH 30 min 1 50 % Epon resin 100 % Epon resin 60 min 2

EponTMresin is used to embed the cell sample before sectioning. The basis is a thermosetting copolymer called epoxy, which forms as the epoxide resin and the polyamine hardener react with each other. Polymerisation on the coverslip was carried out at 65 "C over night. The coverslip was removed by dipping the blocks of partially polymerised resin into liquid nitrogen, which causes the coverslip to break. The glass was carefully removed after warming the block back to RT, with the cells remaining in the resin block. Full polymerisation of the resin was achieved by incubating the blocks for another two days at 65 "C.

2.2.4.2 Sectioning and Staining

Blocks were sectioned by first trimming them around a region of interest (determined with the help of a light microscope) to a surface of around 0.5 x 0.5 mm at the face of the cells. The trimmed pieces were then sectioned along the surface to generate 70 nm thick sections, which were collected on copper grids for staining.

Sections for electron microscopy were stained with lead and uranium. All solutions were prepared as little drops on parafilm and copper grids with sections were placed onto the drops for the required incubation time. Firstly, while carefully protecting the solution from light, uranyl acetate (2 % from

4 Oh stock solution) was prepared in 48 % EtOH. The grids were placed onto the solution for 3 min at RT and then washed three times in water drops. The lead staining solution was prepared by mixing 2.1 mL lead nitrate (189 mM) and 2.1 mL sodium citrate (294 mM) with 800 pL NaOH (0.16 N) while avoiding exposure to C02. Drops of lead stain were prepared in a glass dish

46 containing KOH pellets and the grids added for 3 min at RT, while opening the dish as little as possible. Grids were washed another three times in water drops and dried carefully. Sections on grids were stored at RT until imaging.

2.2.5 Plasmids and Transfection

Transient transfection of primary astrocyte cultures was achieved with the aid of the commercially available transfection reagent Lipofectamine 2000. This cationic lipid forms complexes with DNA, which are internalised by the cells. High transfection efficiency can be achieved at low toxicity.

2.2.5.1 Cell Transfection

For transfection experiments, astrocytes were seeded into 24-well plates containing coated coverslips as described above (pg. 38). One day after seeding, the cells were washed three times in sterile PBS and 300 pL transfection DMEM (containing only Q and FBS) were added per well. The transfection mix was prepared by mixing DNA and Lipofectamine 2000 with Optimem each and incubating both solutions for 5 min at RT. The DNA and Lipofectamine solutions were then thoroughly mixed and incubated at RT for 20 min. The transfection mix was carefully added to the wells drop by drop to ensure even distribution and proper mixing with the medium. For every well of a 24-well plate 1 pg of DNA was mixed with 2 pL Lipofectamine in 25 pL Optimem each. The total volume of transfection mix per well was 50 pL. The cells were incubated at 37 "C for 8 h before replacing the transfection medium with full DMEM to allow for cell recovery over night. Angiogenin treatment of transfected astrocytes was performed as described above (PS.40)- 2.2.5.2 Plasmid Amplification

To amplify bacterial plasmids, competent E. coli bacteria (DH5a or Machl) were transformed with 100 ng of plasmid DNA. The DNA was added to 50 pL thawed bacteria on ice for 20 min. The bacteria were subjected to heat shock for 1.5 min at 42 "C, placed back on ice for 2 min and 450 pL of prewarmed LB (Luria Burtani) broth were added. After 30 min of recovery at 37 "C while shaking, the bacteria were plated on the appropriate ampicillin- or kanamycin-containing LB agar plates (as indicated below) and incubated over night at 37 "C upside down.

An individual colony was picked from the plate and put into 4 mL fresh LB broth containing the appropriate antibiotic. The bacteria were left to grow at 37 "C shaking vigorously for 6 to 8 h until the starter culture was turbid. An over-night culture was then prepared from this starter culture by adding 200 pL of bacteria to 200 mL LB broth containing the appropriate antibiotic. After this culture was left to grow at 37 "C over night shaking vigorously, the bacteria were cooled on ice and centrifuged at 6000 g for 10 min at 4 "C to pellet the bacteria. The supernatant was discarded and the bacterial pellet frozen at -20 "C until plasmid preparation.

Plasmid DNA was extracted using the Qiagen Endofree Plasmid Purification Kit. Pellets were resuspended in PI buffer, making sure no bacterial lumps remained. Lysis buffer P2 was added, the solutions mixed thoroughly and incubated at RT for 5 min. Lysis was stopped by adding precooled buffer P3 and mixing thoroughly. The lysate was poured into a filter cartridge and incubated at RT for 10 min. After filtering the lysate, 2.5 mL buffer ER were added and mixed thoroughly. To remove bacterial endotoxins, which can interfere with plasmid transfection, the solution was incubated for 30 min on ice. During incubation, the columns were equilibrated with buffer QBT. The lysate was added to the column and allowed to pass by gravity flow allowing for DNA binding to the column. It was washed twice in QC buffer and the DNA eluted from the column using buffer QN. The DNA was precipitated with isopropanol at RT and the DNA pelleted at 5,000 g for 60 min at 4 "C. After decanting the supernatant, the pellet was washed with 70 % EtOH and centrifuged again at 5,000 g for 60 min at 4 "C. The supernatant was discarded and the pellet dried for 5 to 10 min.

The pellet was resuspended in TE buffer and the DNA concentration determined using a cuvette-free spectrophotometer. The absorbance of the sample was measured at 260 nm and 280 nm against the diluent as a blank, and the DNA concentration calculated as follows:

Absorbance 260 nm X dilution factor X 50 (const.) = pg/mL plasmid DNA

DNA absorbs at 260 nm and other contaminating compounds and proteins absorb at 280 nm, accordingly the ratio between the two absorbance measurements can be used as an estimate of DNA purity and should be around I.8. Plasmid DNA was stored at -20 "C until use.

2.2.5.3 Protein Overexpression

All overexpressed proteins were tagged with GFP to be able to estimate transfection efficiency and to easily distinguish transfected and untransfected cells. Accordingly, appropriate control cells are present within the same well. The following vectors were used to elucidate endocytosis of angiogenin in astrocytes:

GFP-tagged rAP180 C-terminus (kind gift of Dr McMahon): The C-terminus of the clathrin adaptor protein AP180, which lacks the membrane anchor but still binds clathrin (Kanamycin resistance)

GFP-tagged dominant-negative dynamin 1 (K44A; Damke et al., 1994): A dynamin mutant that cannot bind GTP and hydrolyse it to drive vesicle fission (Ampicillin resistance)

turboGFP-tagged syndecan 4 (MG201975; OriGene, Ampicillin resistance)

Control plasmid: plRES-eGFP (6064-1 ; Clontech; Ampicillin resistance) For overexpression experiments, successful transfection was confirmed 8 h after transfection and angiogenin treatment was carried out 24 h after transfection.

2.2.5.4 Protein Knock Down

Protein expression can be suppressed by RNA interference, a pathway that was first described in plants (Hamilton and Baulcombe, 1999). Presumably as part of normal antiviral defence, long double-stranded RNAs entering the cell are recognised and cleaved by dicer, resulting in the production of so- called small interfering RNA (siRNA). Single-stranded siRNA binds cellular proteins forming the RNA-induced silencing complex (RISC), which degrades one strand of the siRNA leaving only the complementary strand in the complex (Martinez et al., 2002). This allows for recognition of complementary mRNAs, which are then cleaved by RISC and completely degraded by the cell. Similar pathways also operate in mammals as part of antiviral defence and triggered by endogenously expressed microRNAs (reviewed by Hannon and Rossi, 2004).

In this study, transfection of short hairpin RNA (shRNA)-expressing plasmids was chosen to silence gene expression. The shRNA expressed from the plasmid forms a hairpin structure by complementary base pairing, which triggers the RNA interference pathway. Complementary mRNA encoding the protein of interest is degraded, leading to suppression of gene expression. Again, to be able to easily distinguish transfected cells, a GFP expression plasmid was chosen. The GFP is expressed from the same plasmid under the control of a different promoter than the shRNA, leading to expression of both, the shRNA and GFP in the same cell.

To knock down syndecan 4 expression, turboGFP-coexpressing shRNA constructs were chosen (TG513122; OriGene, Kanamycin resistance). The kit contains four specific shRNA constructs, the control turboGFP plasmid and a scrambled sequence plasmid as a negative control. Following transfection, the astrocytes progressively detach from the coverslip. To obtain sufficient protein knock down while preserving as many cells as 50 possible, angiogenin treatment of shRNA-transfected astrocytes was performed 48 h after transfection.

2.2.6 Western Blotting

Quantification of protein expression can be performed by Western blotting. Total cell lysate of the cells of interest is collected and subjected to SDS gel electrophoresis as described by Lammli (1 970). Sample preparation requires for reduction of disulfide bonds within the proteins, followed by protein denaturing with SDS at high temperatures. The resulting negatively charged SDS-protein-complexes with a constant weight-to-charge ratio are then separated on an SDS polyacrylamide gel. Discontinuous SDS Polyacryl- amide Gel Electrophoresis (PAGE) was performed to achieve protein separation into well defined bands. This method requires for the use of a stacking gel (pH 6.8) with wide pores on top of a resolving gel (pH 8.8) with small pores. At the transition between the two gels, the proteins are slowed down due to the difference in pH and pore size between the gels, leading to well defined bands, as all proteins start from the same line through the actual resolving gel. The acrylamide percentage of the gel can be adjusted for optimal separation, higher percentages for smaller proteins and lower percentages for large proteins.

Separated proteins can be transferred onto a nitrocellulose or PVDF membrane by semi-dry transfer and detected using appropriate primary antibodies, which specifically recognise the protein bound to the membrane. Signal detection is usually performed using horse radish peroxidase (HRP)- coupled secondary antibodies raised against the species of origin of the primary antibody. The enzyme converts a substrate into a luminescent product in a concentration-dependent manner allowing for comparison of the signal intensity between bands. The luminescence can be detected by using conventional photographic film or digital imaging systems. 2.2.6.1 Sample Preparation

Cells were treated with angiogenin and endocytosis inhibitors as described above (pg. 40). Lysis buffers (Table 5) were prepared freshly by adding protease and phosphatase inhibitors and the samples were kept on ice during collection. After three washes in PBS, the cells were collected with the help of a cell scraper in 200 pL lysis buffer per well of a 6-well plate (100 pL for 12-well plates).

Table 5: Lysis buffers SDS lysis buffer RlPA lysis buffer 1 RlPA lysis buffer 2

2 % SDS 0.1 % SDS 0.1 %SDS 62.5 rnM TrisIHCI pH 6.8 50 rnM Tris pH 8 50 rnM Tris pH7.5 10 % Glycerin 1 % NP40 1 % Triton XI00 150 rnM NaCl 150 mM NaCl 1 mM EDTA 2 rnM EDTA 0.5 % Sodium deoxycholate 2 mM EGTA 5 mM DTT

Protein content of cell lysates was determined using a bicinchoninic acid (BCA) protein determination kit. Protein peptide bonds reduce CU*+ to CU", which forms a purple compound with the bicinchoninic acid. The absorption of the product can be measured at 562 nm. The kit contains three solutions A, B and C, which are mixed at a ratio of 25 125 1 1 to prepare the detection solution. The assay was performed in a clear flat-bottomed 96-well plate. Each well was first filled with 150 pL of 0.9 % (wlv) NaCl and protein samples (2 pL per well) and BSA standard (0 - 12 pg) were added for duplicate measurements. The plate was incubated for 30 min in the dark at RT, after adding 150 pL of the detection solution. The absorbance was then read at 560 nm in a Tecan plate reader and the protein concentration was calculated by first subtracting the blank (0 pg BSA) value from the BSA standard values. The resulting standard curve was plotted and a regression line calculated. This allows for determination of the protein content within the samples by comparing the averaged, measured absorbance to the standard BSA curve. Protein concentration was adjusted between the samples to load equal protein amounts (20 to 30 pg per well depending on the experiment) and equal volumes to allow for comparison of protein amount between the lanes and to ensure even migration of all lanes. In preparation for migration and to ensure protein stability, 6 X Lammli loading buffer to make 1 X solutions was added and samples stored at -20 "C until loading (Table 9; pg. 56).

2.2.6.2 Gel Preparation

For angiogenin Western blotting and coimmunoprecipitation, a Mini- PROTEAN Tetra Electrophoresis system was used to prepare gels. Glass plates were carefully washed with water and dried with 70 % EtOH to remove detergent from the surface and assembled. For 1.5 mm spacers, a volume of 8 mL of 15 % resolving gel was prepared according to Table 6, while APS (ammonium persulfate) and TEMED (tetramethylethylenediamine) were added immediately prior to pouring the gel to avoid polymerisation in the tube. The gel was poured between the two glass plates leaving enough room for the stacking gel. lsopropanol was added on top of the liquid gel to remove bubbles along the surface and to prevent the gel from drying while it was setting. After 20 min, the isopropanol was removed by decanting, the gel surface washed six times with dH20 to remove remaining alcohol and residual water was removed using tissue. The stacking gel was poured, a 10- or 15-well comb inserted and the gel was left to set at RT.

Table 6: Gel recipes Resolving gel 15 % Stacking gel

Acrylamidelbis-acrylamide 40 % 3.75 mL 625 p~ Tris 3.75 mL (pH 8.8) 600 pL (pH 6.8) Water 2.3 m~ 3.725 m~ APS 10 % 100 pL 50 pL SDS 10 % 100 pL 50 pL TEMED 100 % 5 PL 2.5 pL 2.2.6.3 Gel Electrophoresis

The comb was carefully removed and the gel was loaded into the electrophoresis system. The anode is at the base of the gel, resulting in the proteins loaded at the top of the gel migrating downwards. The gel tank was filled with tris-glycine electrophoresis buffer (Table 9; pg. 56). The protein samples in Lammli buffer were boiled at 95 "C for 5 min to denature the proteins and carefully loaded into the wells avoiding sample carry-over into neighbouring wells. The first well was loaded with 4 pL of protein ladder (adjusted to the same volume as the samples) to estimate the molecular weight of the proteins separated. The gel was run at 60 mA until the proteins reached the resolving gel. Protein separation was petformed at 85 mA for around 90 min or until the migration front reached the edge of the gel.

2.2.6.4 Semi-Dry Transfer

During electrophoretic protein separation, nitrocellulose membranes and Whatman paper were prepared to fit the size of the gel. Whatman paper and nitrocellulose were soaked in transfer buffer I (Table 9; pg. 56) for 5 min. The transfer cell was cleaned with EtOH, a first layer of Whatman paper was put into the cell and bubbles carefully removed. The nitrocellulose was placed on top of the Whatman paper and the gel was removed from the tank. The two glass plates were carefully separated and the stacking gel removed. After soaking the gel in transfer buffer, it was placed on top of the nitrocellulose membrane and covered with another layer of Whatman paper. All bubbles were carefully removed and the lid was put onto the transfer cell. The transfer was carried out at 18 V for 1.5 h.

After the transfer, nitrocellulose membranes were washed three times in dH20 and immersed in Ponceau to confirm efficient transfer and even protein loading. Before recording the Ponceau staining with a digital imaging system, superfluous stain was removed by rinsing the membrane in dHnO, which does not destain the membrane due to the lack of ions. 2.2.6.5 lmmunoblotting

In order to remove all remaining Ponceau stain, the membranes were washed with TBS-T (0.1 % Tween 20) three times, followed by blocking in milk or BSA in TBS-T, depending on the primary antibody used (Table 7). Blocking was necessary to reduce unspecific interaction of the primary antibodies with the membrane. Membranes were incubated with primary antibodies in TBS-T over night at 4 "C while shaking. After three washes in TBS-T for 15 min each at RT, the nitrocellulose membranes were incubated with appropriate HRP-conjugated secondary antibodies (Table 8) diluted Iin 5,000 in TBS-T for 1 h at RT. Following thorough washing in TBS-T (four times; 15 min), protein bands on membranes were visualised using enhanced chemiluminescent (ECL) substrate solution. The ECL substrate was prepared freshly by mixing the two reagents at the recommended ratio. Excess liquid was removed from the membrane and the substrate added onto the nitrocellulose membrane. The chemiluminescent signal was detected in an LAS 3000 Reader and recorded at several exposure times depending on signal intensity. An image of the protein ladder was taken to determine protein size on the membrane.

Table 7: Primary antibodies for Western blotting Antigen Catalogue no Origin Manufacturer Dilution Blocking

Anti p-actin Mouse Sigma 5 % milk Anti pan actin Mouse NeoMarkers 5 % milk Anti angiogenin Goat Merck 5 % milk Anti collagen IV Rabbit Abcarn 5 % milk Anti histone H4 Rabbit Abcarn 5 % milk Anti lLlra Goat R&D Systems 5 % milk Anti MMP3 Rabbit Abcarn 5 % milk Anti Plau Rabbit Abcarn 5 % milk Anti Srsfl Mouse Santa Cruz 5 % milk Anti syndecan 4 Rabbit Abcarn 3 % BSA Anti TDP43 Rabbit Abcarn 5 % milk Anti Tgfbi Rabbit Abcarn 5 % milk Anti tubulin Mouse Sigma 5 % milk Table 8: Secondary antibodies for Western blotting Anti-serum Catalogue Origin Conjugate Manufacturer Dilution no Anti goat API O6P Donkey HRP conjugate Millipore 115000 Anti rabbit AP132P Goat HRP conjugate Millipore 115000 Anti mouse AP124P Goat HRP conjugate Millipore 115000

Table 9: Buffers for Western blotting Lammli loading Electrophoresis Transfer- Transfer- TBS buffer 6 X buffer pH 8.3 buffer I buffer 2 pH 7.4 pH 8.3 0.35 M Tris pH6.8 25 mM Tris 25 mM Tris 48 mM Tris 200 mM NaCl 30 % Glycerol 250 rnM Glycine 192 mM 39 mM 50 mM Tris Glycine Glycine 10 % SDS 0.2 % SDS 20 % Methanol 20 % EtOH 0.6 M DTT 0.012 % Bromophenol Blue

2.2.6.6 Western Blotting for SILAC Evaluation

Western blotting of conditioned medium samples and whole cell lysates of the corresponding flasks was performed by collecting the supernatant and cells after treatment according to the same protocol as described for SILAC- labelled cultures (see pg. 63). The conditioned medium was centrifuged to remove cell debris and precipitated with Trichloroacetic acid (TCA) as described in detail below (pg. 64). For cell lysis, RlPA buffer 2 was freshly prepared (Table 5; pg. 52) and protease and phosphatase inhibitors added (1 mM sodium orthovanadate; 50 mM NaF; 5 mM sodium pyrophosphate; 25 mM P-glycerophosphate). The cells were collected by washing the flask once with PBS, adding 2 mL RlPA buffer 2 per flask and scraping the cells off the bottom of the flask using a cell scraper. The lysate was repeatedly pipetted up and down to ensure complete cell lysis and collected. After 20 min incubation on ice, the lysate was spun at 13,200 rpm for 15 min at 4 "C. The insoluble pellet containing DNA and membranes, was carefully removed from the tube. Before freezing at -80 "C, Lammli loading buffer was added. The precipitated protein was resuspended in 65 pL 1 X Lammli loading buffer as described for the proteomic gels (see pg 64). Both, cell lysates and precipitates, were boiled at 95 "C for 5 min before loading. Proteins were separated on precast 4-12 % Bis-/Trispolyacrylamide gels in NuPAGE running buffer to ensure optimal protein resolution within a wide range of protein sizes and to increase reproducibility, avoiding the effects of incomplete gel polymerisation. The gel tank was filled with 800 mL IX NuPAGE running buffer (from 20 X stock solution), the gels removed from their packaging and placed into the tank. The gels were loaded with 5 pL protein ladder in a total volume of 15 pL at the center and equal volumes of conditioned medium samples loaded to its left, while equal volumes of lysates were loaded to its right. Separating the conditioned medium from the lysates by the ladder was done to prevent samples of different origin interfering with migration in neighbouring wells. Gels were run at 80 V for 30 min followed by 200 V for 45 to 50 min making sure the migration front reached the very bottom of the gel to catch small proteins.

After protein separation, the samples were transferred onto nitrocellulose membranes by semi-dry transfer as described above, using transfer buffer 2 (Table 9; pg. 56) for 40 min at 20 V. Complete transfer and loading were confirmed by Ponceau staining and immunoblotting was performed as described above (pg. 54).

Signals were detected using ECL substrate and recorded using photographic film at different exposure times.

2.2.7 Coimmunoprecipitation

To identify protein-protein interactions in vitro, coimmunoprecipitation can be used. If two or more proteins form a complex within a cell or solution, an antibody specifically targeting one of the proteins involved can be used to separate the entire complex from the other proteins present in the sample. Agarose beads coupled to protein AIG recognise the antibody and can pull

57 the protein complex into the pellet by centrifugation. Protein A is a protein expressed by the bacterium S. aureus to catch antibodies of the host defence by their constant Fc regions (IgG). Protein NG is a combination of two such proteins to increase its pH stability. The pellet can then be analysed by Western blotting, leading to separation of the constituent proteins of the complex, and probed for the presence of the second protein of interest with an appropriate antibody. During the precipitation, it is important to keep the samples at 4 "C at all times to avoid disrupting the interaction of the antibody with the complex and the protein NGagarose beads.

2.2.7.1 Astrocyte Treatment

Primary astrocytes were seeded into 6-well plates as described above (pg. 38). After two days in culture, the cells were washed three times with sterile PBS and 1 mL fresh Neurobasal (containing PIS and Q) was added per well. The cells were treated with 1 pglmL rhAng for 30 min. at 37 "C followed by 2 h at 4 "C or just 2 h at 4 "C. The incubation at 37 "C was included to allow for internalisation of some of the bound angiogenin at the cell surface. Incubation at 4 "C was chosen to ensure specificity of the binding between angiogenin and its receptor complex.

2.2.7.2 Sample Collection

Following angiogenin treatment, the cells were washed three times in PBS and 500 pL NP-40 lysis buffer were added to each well.

NP-40 lysis buffer

Tris pH7.5 20 mM NaCl 78 mM NP 40 0.5 % Glycerol 10 % EDTA 2 mM DTT 0.5 mM Protease inhibitors Cells were collected using a cell scraper and lysates pipetted up and down several times to ensure complete cell lysis. Following 30 min shaking at 4 "C the protein content was determined using a BCA protein determination kit. Each sample was incubated with 5 pg goat-a-angiogenin primary antibody or control goat-a-Rbx2 (sc-5203; Santa Cruz) antibody over night at 4 "C while shaking. Protein AIG PLUS-Agarose beads were washed three times with lysis buffer by spinning the beads down at 1000 g for 2 min and resuspending them. A bed volume of 30 pL of beads in a total volume of 100 pL in lysis buffer was added to the samples for 4 h at 4 "C while shaking to catch the immuncomplexes. Samples were washed four times in NP-40 lysis buffer and collected in 2 X Lammli buffer for protein separation using 15 % polyacrylamide gels. By adding Lammli buffer and boiling the samples as part of the usual Western blotting routine, protein complexes dissociate into their constituent proteins and the antibody.

2.2.8 Proximity Ligation Assay

Another technique developed by a Swedish company called Olink (www.olink.com), allows for in situ detection of protein interactions by Proximity Ligation Assay (PLA; Soderberg et al., 2006). The assay requires for the use of two antibodies, raised in different species, specific for the two proteins of interest, which should ideally work well together in immunocytochemistry. Cells are prepared like for immunocytochemistry, i.e. treated, fixed, blocked and incubated with primary antibodies. Instead of using fluorescently-labelled secondary antibodies, one uses species-specific secondary antibodies attached to a short DNA strand. Antibodies can be attached to two complementary DNA sequences (referred to as PLA probes PLUS and MINUS) and the appropriate combination of secondary antibodies has to be chosen according to the choice of primary antibodies. If the two secondary antibodies are in close enough proximity (< 40 nm) in the sample tested, they can hybridise via connector oligonucleotides and form a circularised oligonucleotide. The circular oligonucleotide has to be ligated to 59 serve as a template for rolling circle amplification. This allows for amplification of the signal from individual interaction points. The DNA is then hybridised with fluorescently-labelled detection probes to be able to visualise protein interaction within the fixed cell in situ.

Add Iwo Duolink PLA probes, each a Men smndary antibodies are in Llgalion creates a complete swcies-s~ecifrcsecondarvantibodv close ~rowimitvlc40 nmL the DNA circularked oliao Mth a shbrt DNA strand, to bind to- strands interact lhybridiztd with me achprinwryantibody added circleforming DNA oligonw cleotidas

Rolling circle wlificatmnprovides Add fluorescent probes to reveal sevsral hun&edfold redicalm of me lmation- andamolificaim evenh DNA circle

Figure 8: Schematic representation of the experimental steps involved in the Proximity Ligation Assay (PLA). Primary antibodies recognise the two proteins of interest and bind secondary antibodies attached to short DNA sequences. In close enough proximity, the two DNA strands can be connected via a connector oligonucleotide. The resulting circular DNA can be ligated and serve as a template for rolling circle amplification. The amplified DNA can be detected with the aid of fluorescently-conjugated DNA probes (www.olink.com).

The samples are then imaged using fluorescent microscopes and the number of spots detected per cell can be quantified. Cell borders can be made visible by phalloidin staining and nuclei with DAPl after the actual proximity ligation assay. Appropriate controls need to be included: combination of the specific

60 antibody (here mouse anti-Ang) against the protein of interest and an unrelated antibody (here rabbit anti-GFAP) raised in the same species as the second antibody of interest (here rabbit anti-Syn 4); untreated cells and a no primary antibody control. This is important to be able to estimate the background ligation happening between the two secondary antibodies and the specificity of the reaction between the two antibodies of interest.

2.2.8.1 Cell Treatment

Astrocytes were seeded in 8-well chamber slides at 100,000 cells per well in 200 pL full DMEM after coating the glass bottom with poly-L-Ornithine. The cells were transfected according to the method described above (pg. 47) using 400 ng DNA and 800 pL Lipofectamine 2000 in 20 pL Optimem per well 24 h after seeding. The astrocytes were treated with 1 pglmL rhAng for I h and fixed in 4 % PFA, as described above (pgs. 40 and 42). Staining with primary antibodies was performed like for conventional immunocytochemistry (see pg. 42).

2.2.8.2 Proximity Ligation Assay - Procedure

All reactions were carried out at 37 "C in a humidity chamber, the reaction volume was 40 pL per well and all washes were performed on a shaker. Instead of using fluorescently-labelled secondary antibodies, PLA probes were diluted 1 in 5 in antibody diluent and the antibody solution was added for 2 h at 37 "C. Hybridisation stock solution containing connector oligonucleotides was diluted 1 in 5 in dHpO and wells were washed twice for 5 min in PBSITriton. Following hybridisation for 15 min at 37 "C, the amplification stock was diluted 1 in 5 in dH20 and the wells washed twice in TBS-T for 2 min. The TBS-T was carefully discarded, the polymerase enzyme added to the amplification solution and mixed properly. Amplification was carried out at 37 "C for 90 min. All following steps were performed while protecting the samples and reagents from light. Detection stock was diluted 1 in 5 in dH20 and after two 2 min washes in TBS-T, the detection solution was added to the wells and incubated for 60 min at 37 "C. The cells were subjected to a series of stringency washes in SSC buffer (150 mM NaCI; 15 mM Na citrate; pH 7.0) for 2 min each: 2 X SSC, 1 X SSC, 0.2 X SSC, 0.02 X SSC.

2.2.8.3 Cell staining

To be able to distinguish individual cells, the samples were stained with phalloidin and DAPI. FITC-labelled phalloidin was diluted 1 in 120 in PBS, added to the wells and incubated for 30 min at RT. The wells were washed once in PBSITriton, three times in PBS and once in dH20, dried and mounted using DAPI-containing mounting medium. To avoid dissociation of the DNA complexes from the detection sites, imaging was performed within the following three days. The samples were stored at 4 "C between imaging sessions. Signals were detected using both a TE 300 epifluorescent microscope and an LSM 710 confocal microscope. Spots were quantified using the Blob finder software (settings: minimal nucleus size I00 pixels, cytoplasm size 100 pixels; blob size 3x3 pixels) provided by Olink Biosciences on epifluorescent images at 60 X magnification. Counts were tested for statistical significance between three separate experiments using Student's t-test.

2.2.9 Sample Preparation for Mass Spectrometry

Sample preparation for quantitative mass spectrometry requires for labelling of the cells with isotope-labelled amino acids. These are not radioactive, so no special precautions are necessary during cell culture. Special care needs to be taken to avoid contaminating the samples with ubiquitous proteins that are found in labs such as serum or milk proteins as well as keratins from the skin or hair. Accordingly, all experiments were performed wearing separate lab coats and gloves, as well as tying back long hair and avoiding woollen clothing on the days of sample preparation. 62 2.2.9.1 Primary Astrocyte Cultures

To ensure good incorporation of the modified amino acids into cell cultures, special DMEM without L-Arginine and L-Lysine needs to be used and the medium is supplemented with dialysed serum, from which all amino acids not part of proteins have been removed. Accordingly, the medium needs to be substituted with these two essential amino acids. Stocks of heavy (ArglO (L-[U-I 3C6-15N41 Arginine) and Lys8 (L-[I 3C6-15N21 Lysine)) and medium (Arg6 (L-[U-13C61 Arginine) and Lys4 (L-[2H4] Lysine)) isotope-labelled amino acids (see pg. 136) were prepared at 1,000 X and aliquotted to make 50 mL of DMEM. For L-Lysine, the stocks were prepared at 147.5 mg1mL and for L-Arginine at 91.3 mglmL to make up DMEM with the ususal 0.1475 g1L L-Lysine and 0.0913 glL L-Arg. Heavy and medium SlLAC DMEM was otherwise supplemented with the usual PIS and Q.

Astrocytes were prepared from P2 C57 Black 6 mouse pups as described above (pg. 38). Instead of pelleting the dissociated cortical cells from 5 or 6 pups in one tube, the cells were first split evenly into two tubes and pelleted separately. The cells were resuspended in either heavy or medium full SlLAC DMEM and plated in T75 tissue culture flasks. After 7 and 13 days in vitro (DIV), the medium was replaced following one wash in sterile PBS. The incorporation of labelled amino acids was first assessed by collecting astrocytes both, after one and two weeks of labelling.

As rhAng treatment needed to be performed in Neurobasal medium containing light L-Lysine and L-Arginine, the experimental protocol was adjusted to optimise amino acid incorporation according to the following table (Table 10).

Table 10: Optimisation of experimental protocol Treatment Protocol 1 Protocol 2 Protocol 3

Medium change at 7 DIV + + + Medium change at 13 DIV - + + NB 6 h + + + Labelled amino acids added to NB - - + DMEM (without serum) 18 h + + + 63 One additional medium change the day before treatment was included to avoid treating the starved cells with rhAng in Neurobasal containing light amino acids. Otherwise, the astrocytes might take up all the light amino acids from the Neurobasal. Additionally, it was tested to supplement normal Neurobasal with the same amounts of L-Lysine and L-Arginine as normally used for preparing SlLAC DMEM, resulting in double the concentration of these two amino acids in the medium. No toxic effects of the two amino acids at these concentrations had been reported, nor were any observed in the astrocyte cultures used for this study. Protocol 2 was chosen for all treatments thereafter, based on the mass spectrometry results obtained (see scheme pg. 143):

On day 14 in vitro, astrocytes were washed 6 times in PBS to remove all serum and treated with 1 vglmL rhAng, 50 pglmL BSA or were left untreated in Neurobasal medium (containing PIS and Q) for 6 h at 37 "C to ensure uptake of angiogenin by astrocytes. Following another two washes in PBS, the astrocytes were covered with 6 mL fresh SlLAC DMEM (containing PIS and Q) for another 18 h.

2.2.9.2 Medium Collection and Protein Precipitation

Conditioned medium was collected and pooled from the treated and control flasks, cell debris pelleted at 1,000 rpm for 5 min and the supernatant precipitated using 10 % TCA on ice for 30 min. Cell lysates were collected as described above for Western blotting (pg. 56) Precipitated proteins were spun down at 4 "C, 13,200 rpm for 20 min and washed three times with diethyl-ether to remove any remaining salt and TCA from the protein pellet. The pellet was dried over night at RT and frozen at -80 "C until protein separation.

2.2.9.3 Protein Separation by Gel Electrophoresis

Protein separation was performed on big 30 mL 12% polyacrylamide gels to separate proteins sufficiently and to obtain clear bands. Firstly, glass plates 64 were cleaned very carefully with 70 % EtOH avoiding contamination of the surface with foreign proteins. Two glass plates separated by spacers on both sides were assembled into the rack provided for casting the gel. To avoid leakage of the gel, the glass plates were sealed on either side and at the bottom edge using I % agarose. The resolving gel (Table II) was poured using a 25 mL pipette, covered with isopropanol and left to set for 1.5 to 2 h at RT. Before pouring the stacking gel (Table II), the isopropanol was decanted, the resolving gel washed six times with Braun water and residual water carefully removed using tissue. Traces of bromophenol blue were added to the stacking gel solution, the gel was poured and the comb carefully fitted. Adding bromophenol blue to the stacking gel facilitated monitoring of the initial gel migration in the big gel tanks used, as migration through the stacking was very slow. The resolving gel had to be prepared using very little APS and less bisacrylamide than commonly used to prevent the gel from becoming too rigid for the extraction of the peptides from the gel after in-gel digestion. To allow for complete polymerisation of the resolving gel and to avoid the formation of polyacrylamide adducts with peptides, it was left to set for at least one night, maximally four nights at 4 "C covered with a wet piece of tissue and parafilm to prevent the gel from drying.

Table 11 : Mass spectrometry gel recipies Resolving gel 12 % Stacking gel

Acrylamide 40 % 9 m~ Bis-acrylamide 2 % 2.37 m~ Tris 7.5 mL (I.5 M; pH 8.8) Braun water 10.76 rn~ APS 10 % 42 p~ SDS 10 % 300 1L TEMED 100 % 15 pL

The gel was prepared for loading by removing the comb and washing the wells with Braun water. The gel was fitted into the electrophoresis tank opposite a buffer dam. The gel tank was filled with four litres of electrophoresis buffer (25 mM Tris, 195 mM Glycine, 0.1 % SDS) covering the whole gel, to ensure the temperature was constant across the length of 65 the gel. The tank was left for 15 min to observe whether the inner chamber was leaking.

Precipitated proteins were resuspended in 30 pL 2 X Lammli buffer for 5 min at 95 "C followed by 1 h shaking at RT to ensure complete resuspension. The sample was loaded at the centre of the gel leaving one well empty between the ladder (I0 pL adjusted to 30 pL total volume) and the sample to avoid spillage in either direction. The empty wells surrounding the ladder and the sample were filled with the same volume of 2 X Lammli buffer to ensure a straight migration front. The gel was run for I h at 25 mA to allow for the proteins to enter the resolving gel and to minimise electroendosmosis, followed by approximately 2 h at 30 mA to separate the proteins over a gel length of about 4 cm.

2.2.9.4 Gel Fixation

Gel fixation solutions were prepared freshly just before stopping electrophoresis using Braun water. The gel was removed from the tank, the two glass plates carefully separated and the gel was fixed in 50 % ethanol containing 5 % acetic acid for 20 min at RT under gentle agitation without removing the stacking gel. After one wash in 50 % ethanol for 10 min and three further washes in Braun water for 5 min each, the gel was stained using PAGE Blue (prewarmed to RT; reused up to three times) over night under gentle agitation at RT. Small crystal precipitates form in the PAGE Blue solution at 4 "C, which damage the gel. Gels were washed twice with Braun water, scanned (300 dpi; 16 bit; grey levels) and 20 lanes assigned for excision.

2.2.9.5 In-Gel Protein Digestion

Gel bands were excised using scalpel blades under a laminar flow hood to prevent contamination with foreign proteins. The gel was placed on a glass plate and kept wet with Braun water throughout the excision procedure. Bands were excised individually starting from the top end of the gel. A fresh 66 scalpel blade was used for every band to avoid protein carry-over. The bands were cut into gel cubes of approximately 1 mm3 and gel cubes from one band were put into a fresh eppendorf tube (1.5 mL polypropylene Click Fit).

All reagents for protein digestion and extraction were prepared freshly, only filtered tips were used in preparation for digestion. The procedure was carried out under laminar flow. The gel cubes were destained once using 200 pL 50 % acetonitrile in 50 mM ammonium bicarbonate for 10 min at room temperature while shaking vigorously, the supernatant was discarded and the gel pieces washed a second time using 500 pL 50 % acetonitrile in 50 mM ammonium bicarbonate. The supernatant was removed using the same tip for all samples, as proteins are tightly bound within the gel until digestion. The gel was dehydrated for 10 min with 200 pL acetonitrile and the supernatant was discarded carefully without aspirating shrunken gel cubes. The gel was rehydrated by adding a volume of 100 mM ammonium bicarbonate just sufficient to cover the gel pieces for 10 min to estimate the minimal digestion volume needed. The same volume of acetonitirile was added for another 10 min. As the gels prepared for this study were of the same size and the banding pattern very similar, a volume of 200 pL was used for rehydration. The supernatant was discarded and gel pieces were dried using a SpeedVac for 3 min. The SpeedVac had to be turned on 45 min prior to use. Following rehydration of the gel pieces in 120 pL of 100 mM ammonium bicarbonate containing 15 ng/pL trypsin for 45 min on ice, the proteins were digested at RT over night while shaking.

2.2.9.6 Peptide Extraction

After in-gel digestion of the proteins, the risk of contamination is reduced, as any foreign protein will remain intact and therefore show at completely different mass to charge ratio (mlz) than the peptides. For the following extraction, it is very important to keep the same tip for every individual sample. Most of the peptides are found in the supernatant straight after digestion. Peptides have a high affinity for the plastic pipette tips commonly used, thus the tip surface will be saturated with peptides during the first removal of supernatant into a fresh tube. The gel pieces were dehydrated by adding 120 pL acetonitrile for 10 rnin and the supernatant collected into the same tube using the same tip for every individual sample originating from one band. The gel was rehydrated for 10 rnin with 120 pL 100 mM ammonium bicarbonate and 120 pL acetonitrile were added for an additional 10 min. The supernatant was collected and two additional extraction steps were performed as follows: Firstly, 120 pL of 5 % formic acid were added for 10 rnin followed by 120 pL acetonitrile for 10 min. The supernatant was collected. Extracted peptides were dried using the SpeedVac for approximately 4 h in preparation for mass spectrometric analysis. Dried samples were frozen at -20 "C until injection into the mass spectrometer.

2.2.9.7 Mass Spectrometry

Detailed explanation of the technical procedures and physical background on mass spectrometry (MS) will be given in 4.1 .I pg 132.

Reversed phase separation of peptides was performed using a U3000 nanoflow HPLC system. Peptides were dissolved in solution A (Table 12) and loaded onto a precolumn (0.3 mm x 10 mm) for 3 rnin to remove any remaining salt present in the sample before gradient elution. Peptides remain attached to the C18 column material in solution A. Peptides were eluted from the precolumn into the capillary (0.075 mm x 150 mm) of a reverse-phase Pepmap@ C18 column and were separated for 60 rnin by progressively increasing solution B from 0 to 40 % at a flow rate of 300 nLlmin. The C18 columns are best suited for separating peptides according to their hydrophobicity as they attach to the column material in aqueous buffers and can be eluted using increasing concentrations of acetonitrile. The separation was followed by a 15 min wash in 80 % solution B (Solutions A and B see Table 12). Eluting peptides were electrosprayed online at a voltage of 2.0 kV into an LTQ Orbitrap Velos mass spectrometer. Throughout the nano liquid chromatrographic separation, the mass spectrometer continuously obtained full-scan mass spectra ranging from 400 to 1600 mlz (mass-to-charge ratio) at a resolution of 60,000 (at 400 mlz) followed by twenty data-dependent MSIMS scans of the twenty most abundant ions. Once a peptide had been recorded, it was no longer taken into account for further MSIMS recording by dynamic exclusion for 20 s. All MSIMS spectra were recorded with normalised collision energy of 35 % at an activation of Q 0.25 and an activation time of 30 ms with an isolation window of 2 Th. Data were acquired using Xcalibur 2.1 software.

Table 12: HPLC elution buffers Solution A Solution B

0.1 % Formic acid 0.1 % Formic acid 2 % Acetonitrile In Acetonitrile In Braun water

2.2.9.8 Data Analysis

The MaxQuant software has been developed to detect isotope patterns and assign SlLAC pairs or triplets resulting from labelling (Cox and Mann, 2008). The software assumes trypsin may have missed up to two cleavage sites, thus maximally three labelled amino acids are contained in one peptide. The ratios between the intensities of assigned pairs are determined and based on the assumption that there is no overall differential regulation, the ratios are normalised. With the aid of peptide charge pairs, those are pairs of peaks generated by the same peptide with different charge after ionisation, mass accuracy is corrected which greatly improves results. Generally, species- specific, true protein sequences and reversed nonsense sequences are used for database search and the top ten matches of the fragmentation spectrum are assigned a peptide score. The probability of false hits can be estimated by calculating the posterior error probability (PEP) based on this score and the peptide length. The PEP is then used to determine the peptide false discovery rate (FDR): Identified peptides are sorted according to their PEP until 1 % false hits (from forward and reverse database) have accumulated. The accepted peptides are assigned to proteins, while peptides shared between two proteins (razor peptides) are associated with the protein 6 9 containing most identified peptides. Protein quantification is performed taking unique and razor peptides into account. The peptide PEP is then used to calculate the protein PEP to assess the protein FDR as mentioned above. Database search also identifies peaks not detected to be present as a SILAC pair, thus ratios are calculated by extrapolating the missing partner in the spectrum. Statistical analysis of the data obtained is performed on log2 protein ratios (median of SILAC peptide ratios belonging to one identified protein) by calculating an outlier significance score referred to as significance A. Alternatively, in samples containing large protein numbers, significance B can be determined, which takes into account that unregulated, highly abundant proteins spread less (Cox and Mann, 2008).

Raw data analysis was performed using the MaxQuant software version 1.I .I .36. Retention time-dependent mass recalibration was applied with the aid of a first search database containing abundant mouse proteins and contaminants as implemented in the Andromeda software (Cox et al., 201 1). Peak lists were searched against the UniProt mouse database release 201 1-04 (www.expasy.ch), as well as 255 frequently observed contaminants and reversed sequences of all database entries. The following settings were applied for searches in recorded mass spectra: Spectra were searched with an initial mass tolerance of 7 ppm (MS) and 0.5 Th (MSIMS). The enzyme specificity was set to trypsin, the enzyme used for protein digestion. Only peptides with maximally two missed cleavages were considered for analysis and the minimal peptide length had to be six amino acids. Maximal peptide length was set to 100 amino acids. The search allowed for variable modification of Methionine by oxidation. Protein quantification was based on the median SILAC ratios of at least two peptides (two valid "ratio counts") in each biological sample. MaxQuant standard settings were used to quantify SILAC triplets with maximally three labelled amino acids allowed per peptide. The software was also used to perform multiple significance testing according to the method introduced by Benjamini and Hochberg (1995), which is the standard statistical protocol used for the type of SILAC analysis performed for this study as introduced with the MaxQuant software (Cox and Mann, 2008). Manual re-examination of analysis results was performed by taking into account results obtained in each individual experiment: Only proteins with more than two peptides identified in two or more experimental repeats were included. Furthermore, individual normalised HIM ratios had to be calculated for at least two experiments, however if more than three peptides were detected in individual experiments, proteins were accepted without calculated normalised HIM ratios. If proteins did not test significant in individual experimental repeats, overall regulation in the same direction was confirmed.

2.2.9.9 Data Treatment and Visualisation

Graphical representation only included proteins with at least one quantification per group and plots were generated using the R statistical programming language (R Development Core Team, 201 1).

Functional annotation and visualisation of the data obtained was performed by first identifying the most representative protein identified by MaxQuant based on detected peptides. The proteinGroups.txt file generated by MaxQuant contains groups of proteins that share the same peptides and was therefore used to extract the leading protein. At the Functional Proteomics Platform (FPP, www.fpp.cnrs.fr) in Montpellier, criteria have been defined (in agreement with the MaxQuant community) and an internal tool written in Perl to automate the protein list retrieval:

The source of the protein knowledgebase (from the UniProt database reviewed Swiss-Prot proteins are preferred over unreviewed TrEMBL entries) and the number of gene ontology terms annotated define the leading protein. Manual confirmation of results is rarely necessary in case of multiple choices.

Firstly, contaminants, reverse hits and proteins without calculated normalised HIM ratios are removed from the proteinGroups.txt file. The proteins with the maximum number of identified peptides ("Peptide Counts (all)" column) are extracted from all identifiers of a protein group ("Proteins" column) into match groups to be able to determine the leading protein in each of these subsets. The FPP tool then permits to (1) retrieve up-to-date mouse gene ontology (GO) annotations that are collected by the Mouse Genome Informatics database resource (MGI, www.informatics.jax.org) and updated daily in the "gene-association.mgi" file; (2) assign corresponding UniProt and MGI identifiers used by MaxQuant and GO annotations, respectively; (3) compute the number of GO terms per protein of the match groups to determine the leading ones. Three different mapping files were used to optimise results (MRK-SwissProt-TrEMBL.rpt (MGI); mouse.xrefs (EMBL-EBI, www.ebi.ac.uk/GOA); idmapping.dat (UniProt, www.uniprot.org)) giving priority to MGI over the other two sources.

Furthermore, the FPP tool performs GO analysis based on the retrieved GO annotations with the aid of an R script using the goTools package (Paquet and Yang, 2007; R version 2.5.1), which is available from the BioConductor project (http://bioconductor.wustl.edu/bioc/html/goTools.html, Gentleman et al., 2004). The TlDS method implemented in the ontocompare function of the R script is used to calculate percentages of proteins assigned to GO annotations. Pie charts are created for the three ontologies Molecular Function, Biological Process and Cellular Component.

All intermediate data processes are recorded in a log file and a copy of the original proteinGroups.txt file is generated with additional columns containing the leading protein, match group proteins and the GO terms for the three GO ontologies (all relative to the leading protein).

The resulting total list of leading proteins was subsequently subjected to GO analysis as described above (source "gene~association.mgi", version 201 1/01/27). The GO annotations were represented in graphically improved pie charts generated in Microsoft Excel. Functional pathway annotation was performed on the total leading protein list with the aid of the DAVID (Database for Annotation, Visualisation and Integrated Discovery) bioinformatics resources website (Huang da et al., 2009b, a) and the top ten overrepresented KEGG pathways (Kyoto Encyclopedia of Genes and Genomes; Kanehisa and Goto, 2000), as tested by a modified Fisher's exact test developed for this application, were represented as bar charts displaying the percentage of identified proteins annotated to each pathway along with the Benjamini-Hochberg FDR (1995). Significantly regulated proteins were 72 subjected to SignalP (Bendtsen et al., 2004a) and SecretomeP (Bendtsen et al., 2004b) analysis. SignalP predicts classical protein secretion for eukaryotes and bacteria and the cleavage sites of signal peptides. SecretomeP recognises features of secreted mammalian proteins that are independent of the pathway used for secretion, thus allowing for predicition of non-classically secreted proteins. Furthermore, the list of regulated proteins and the upregulated and downregulated protein lists were subjected to GO analysis using BiNGO 2.44 (Maere et al., 2005) and results were visualised using Cytoscape 2.8.1 (Shannon et al., 2003) to determine overrepresented functional annotations subject to regulation by angiogenin compared to the whole mouse genome. The default BiNGO mouse annotation was used for analysis with the GOSlim-Generic annotation. Statistical overrepresentation (p c 0.01) was calculated with the aid of BiNGO by hypergeometric analysis and Benjamini-Hochberg FDR correction (1995). 3. Chapter I

Angiogenin Internalisation into Astrocytes

3.1 Chapter IA

Angiogenin Uptake into Vesicles in Astrocytes

3. I.I Introduction

3.1 .I.I lntracellular Vesicles

Angiogenin binds a 170 kDa cell surface receptor on endothelial and tumour cells (Hu et al., 1997) as well as cell surface actin (Hu et al., 1993). Angiogenin is then internalised and translocates to the nucleus (Moroianu and Riordan, 1994a; Tsuji et al., 2005), as has been shown for a number of other growth factors (reviewed by Belting, 2003; and Olsnes et al., 2003). Commonly, endocytic events involve sorting of the internalised proteins in vesicular compartments depending on their fate as determined by the receptor and the protein complex assembled around it (Figure 9): Proteins can be destined for degradation in lysosomes, a process that involves acidification of the vesicle, which causes dissociation of the ligand-receptor complex. The receptor may be targeted for recycling back to the plasma membrane or degraded along with the ligand. Alternatively, the internalised protein may likewise be recycled to the plasma membrane or undergo transcytosis, a very important process for selective delivery of molecules across barriers such as the blood brain barrier (reviewed by Wolburg et al., 2009). Accordingly, the two processes of endo- and exocytosis are linked and can be referred to as the endocytic-exocytic cycle (summarised by Lodish, 2000; and Alberts, 2002; and Kittler and Moss, 2006). Figure 9: Endocytosed ligand- bound receptor proteins are sorted along either of three major routes. The receptor is first sorted into early endosomes from where it can be recycled back to the plasma membrane or sorted into lysosomes leading to protein degradation. In polarised cells, the receptor can also be transcytosed to the other membrane face of the cell and any ligands bound are released again (Alberts, 2002).

The endosomal system is comprised of complex, heterogenous compartments of variable shapes and sizes. Endosomes mature after internalisation from more tubular, early endosomes located at the periphery of the cell to spherical, late endosomes. These are found closer to the nucleus of the cell following transport along the cytoskeleton aided by motor proteins towards the centre of the cell during the sorting procedure. The pH value of endosomes is progressively lowered while sorting to allow for lysosomal proteases to degrade endosomal cargo upon fusion (summarised by Lodish, 2000; and Alberts, 2002; and Kittler and Moss, 2006).

3.1 .I.2 Endosomal Markers

Endosomal sorting is tightly regulated and a plethora of proteins involved have been identified. One of the best studied groups of proteins in regulating membrane trafficking are the Rab GTPases, which are members of the ras superfamily of small GTPases. It has been observed that specific Rab proteins are associated with particular subcompartments of the endsomal system (Figure 10). Rab GTPases are found in two states, either membrane- bound or in the cytosol, depending on their GDPIGTP-loading status. Active small GTPases may directly affect membrane curvature, alternatively they can regulate the cytoskeleton or protein-or lipid-modifying enzymes, which in 75 their turn activate and recruit membrane curvature-sensing and -generating proteins. Early endosomes are classically associated with Rab 5, which binds and recruits effector proteins such as early endosomal antigen I by generating phophatidylinositol-3-phosphate on the vesicular membrane. Rab 11 and 4 are found on recycling endosomes, while Rab 11 marks slow recycling involving multivesicular bodies and Rab 4 is associated with rapid recycling straight from the early endosome. The small GTPases Rab 9 and 7 are both associated with late endosomes: Sorting of these back to the trans- Golgi network depends on Rab 9, while Rab 7 regulates fusion with lysosomes (reviewed by Kittler and Moss, 2006; and Doherty and McMahon, 2009; and Grant and Donaldson, 2009).

Figure 10: Schematic overview of sorting steps involved upon vesicle endocytosis with Rab GTPases involved. Clathrin-coated or uncoated vesicles with cargo undergo fission mediated by dynamin. Coat proteins are shed and the vesicle fuses with sorting endosomes with the aid of Rab 5. Cargo proteins are recycled back to the plasma membrane via recycling endosomes (R.E.) in a process mediated by Rab 4 or Rab 11. Alternatively, cargo can be sorted via late endosomes or multivesicular bodies (MVB) to either the Golgi apparatus depending on Rab 9 or it can be fated for degradation in lysosomes requiring Rab 7 (Kittler and Moss, 2006). Non-classical endocytic mechanisms not involving clathrin or caveolin have been shown to be important for internalisation of diverse cargo molecules and some types of cargo may employ distinct endocytic mechanisms in a context-dependent manner. Flotillin proteins display homology to caveolin but are found in distict membrane microdomains and Flotillin 1 has been found to be required for endocytosis of cell surface proteoglycans (reviewed by Doherty and McMahon, 2009).

3.1 .I.3 Studying Vesicular Compartments

To study endosomal sorting the small GTPases discussed above are commonly used to distinguish individual sorting steps and compartments and to retrace the route of endocytic cargo within cells. However, different cell types may employ small GTPases in different endocytic pathways, making this approach not very specific. Nevertheless, it has proven to be very useful to study endocytosis with the help of these markers (reviewed by Doherty and McMahon, 2009). Many antibodies are commercially available to detect Rab GTPases and other proteins involved in sorting of endosomes.

Alternatively, fluorescent approaches have been developed to monitor endocytic events by live cell imaging. The uptake of fluorescently-labelled transferrin is probably one of the best studied examples (reviewed by Doherty and McMahon, 2009). In addition to labelling cargo or receptor, fluorescent probes can be used which specifically label distinct cellular organelles. Probes for all major cellular organelles such as mitochondria, the ER, the Golgi apparatus or acidic organelles like lysosomes are commercially available with different fluorescent spectra to allow for combinatorial labelling. These probes have been developed to be cell permeable and can thus be used for live cell imaging, but with appropriate fixation methods they can be employed for immunocytochemical colabelling, too. Specific accumulation of the probes in different organelles is achieved by exploiting the microenvironment of the organelle (e.g. acidic pH in lysosomes) or by labelling specific proteins or lipids enriched in particular organelles (e.g. potassium channels in the ER; LifeTechnologies, 201 0). The techniques described above make it possible to follow the endocytic route taken by internalised cargo proteins by cell imaging approaches and were therefore used to gain insights into angiogenin sorting following internalisation.

3.I .2 Results

3.1.2.1 Cell Type-Specific Angiogenin Internalisation

It had previously been observed that motoneurons show strong basal angiogenin expression (Greenway et al., 2006) and recombinant human angiogenin protein protects motoneurons in primary mixed motoneuron cultures derived from El3 mouse embryos from toxicity induced by a-amino- 3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) and ER-stress (Kieran et al., 2008; Sebastia et al., 2009). To further elucidate the mechanism responsible for angiogenin's neuroprotective properties, primary mixed motoneuron cultures were treated with 1 pg/mL rhAng for 1 h at 7 DIV, fixed and stained for angiogenin as well as the astroglial marker GFAP or the neuronal marker NeuN (NEUronal Nuclei). This revealed that glial cells surrounding neurons take up recombinant human angiogenin into cytoplasmic vesicles (Figure 11; arrow heads), whereas neuronal cells showed diffuse staining of the cell cytoplasm excluding the nucleus. (Figure 11; left panel). Costaining with GFAP revealed that astroglia contain many angiogenin-positive vesicles after treatment (Figure 11, right panel). GFAP is an intermediate filament protein abundantly expressed by astrocytes, therefore staining for GFAP allowed for visualisation of cell morphology. I NeuN

0 DAP l DAPl

NeuN Ang DAPl

Figure 11: Recombinant human angiogenin applied to primary mixed motoneuron cultures is endocytosed by the glial cell population. At DIV7, primary mixed motoneuron cultures were treated with rhAng (1 vg/mL) for 1 h prior to fixation and staining for angiogenin (red) and the neuronal marker NeuN (green; left panel) or the astroglial marker GFAP (green; right panel). Arrow heads indicate angiogenin containing vesicles. Depicted are representative images from two experiments performed with similar results. Scale bar 25 pm. Primary mixed motoneuron cultures also contain many glial cells surrounding the neurons such as astrocytes, fibroblasts, endothelial cells and microglia, the macrophages of the CNS. As motoneuron cultures are very difficult to obtain and therefore very precious, all further studies to characterise angiogenin uptake were carried out using primary astrocyte cultures derived from the cortices of P2 mouse pups. These cultures were more readily available and contained a higher percentage of astrocytes (76 % on average across all experiments counted). Cortical cultures still contained some contaminating microglia and fibroblasts and treating these cultures with rhAng for 1 h revealed the same pattern of vesicular angiogenin staining as observed in the glial population of cells in primary mixed motoneuron cultures (Figure 12). To find out if this staining pattern was specific for astrocytes or microglia, colabelling with GFAP or the microglial marker CDllb was performed. Like in primary mixed motoneuron cultures, GFAP-positive astroglia showed punctate angiogenin staining (Figure 12; arrow heads; left panel). Similarly, CDl Ib-positive microglia displayed vesicular uptake of angiogenin (Figure 12; 3rd panel) and as CDl Ib is involved in adhesion of microglia, staining revealed the exact attachment zone of individual cells. Only very low basal expression of angiogenin was observed in both cell types (Figure 12; 2" and 4th panel).

Further endocytosis studies were performed on primary astrocyte cultures costaining only for GFAP. As the staining pattern for microglia was so similar, it cannot be excluded that angiogenin uptake into microglia may follow a similar route. Ang no Ang Ang no Ang I.

1 Ang DAPl

Figure 12: Recombinant human angiogenin applied to primary cortical astrocyte cultures is endocytosed by both astrocytes and microglia. Primary P2 cortical astrocyte cultures were treated with rhAng (1 vg/mL) for 1 h prior to fixation and staining for angiogenin (red) and the astroglial marker GFAP (green; left panel) or the microglial marker CDl Ib (green; right panel). Arrow heads indicate angiogenin containing vesicles. Depicted are representative images from two experiments performed with similar results. Scale bar 25 pm. 3.1.2.2 Astrocyte Morphology

Astrocyte morphology in primary cultures was examined in more detail using transmission electron microscopy, to be able to characterise the cells further and to gain insights into the vesicular structures present. The technique used for this experiment allows for conservation of fine structure as the cells are fixed on the glass coverslip, which is later carefully removed. It has previously been observed that adult astrocytes abundantly express intermediate filaments, mainly GFAP and vimentin, in a resting state (Eliasson et al., 1999). Upon activation they additionally express nestin and upregulate the expression of GFAP and vimentin. In electron microscopy, abundant intermediate filament (IF) expression was observed as indicated (Figure 13A) as well as prominent microtubules (MT). Thus, with the aid of intermediate filaments astrocytes were distinguished from contaminating microglia and fibroblasts. Besides expected vesicular organelles like the Golgi apparatus (Go), the rough endoplasmic reticulum (rER), mitochondria (Mi) and plenty of ribosomes (Ri), the rugged structure of the cell surface of astrocytes was revealed in more detail (Figure 13A). Astrocytes showed many filopodial processes and abundant vesicles (Ves) especially close to the plasma membrane (Figure 13B). The network of intermediate filaments was very dense at the edges of the cells and vesicles were observed at close proximity of microtubules (Figure 13C). Figure 13: Overview of astrocyte morphology in primary cortical cultures by transmission electron microscopy. Primary P2 cortical astrocyte cultures were fixed for electron microscopy, embedded, sectioned and stained. A) Images show intermediate filament (IF)-rich astrocytes with all elements of eukaryotic cells: Go: Golgi apparatus; Nc: Nucleus; Mi: Mitochondria; rER: rough Endoplasmic reticulum; MT: Microtubules; Ri: Ribosomes. Astrocytes in cell culture also contain many vesicles (Ves) and granules (Gr). B and C) Details of astrocyte structure at cell borders with vesicles and elements of the cytoskeleton densely packed. Following the initial observation that angiogenin was taken up into vesicular structures in astrocytes, particular attention was paid to organelles resembling vesicles. A characteristic feature of the astrocytes in primary culture was the presence of large, dark granules (Gr) throughout the cytoplasm (Figure 14B). The fine structure of these granules was reminiscent of rnyelin sheaths surrounding the axons of neurons and could be remains from the tissue preparation or autophagic vacuoles (Figure 14A and C; arrow heads indicate membrane layers). At the cell surface, coated pits (CP) were observed (Figure 14A), as well as coated vesicles (CV) and those that were just shedding their protein coat (Figure 14D; left vesicle). Astrocytes also contained the usual sorting stations for proteins to be secreted (rough endoplasmic reticulum (rER) and Golgi apparatus (Go); Figure 14G and H), as well as those that have been endocytosed, like multivesicular bodies (MVB; Figure 14F). Interestingly, multiple cilia were observed in astrocytes in primary cell culture (Figure 141 and J). Most cell types normally express one primary cilium, which has been described to be involved in signalling (reviewed by Gerdes et al., 2009; Yoshimura et al., 201 1). Multiple cilia on astrocytes have been observed in the aging subventricular zone of mice (Luo et al., 2006), however in the current study they might represent artefacts due to cell culture conditions. Figure 14: Detailed view of astrocyte morphology in primary cortical cultures by transmission electron microscopy. A) Granule (Gr) in astrocytes with multiple layers of membranes surrounded by intermediate filaments (IF) and mitochondria (Mi) in close proximity of a coated pit (CP) at the plasma membrane. B) Overview of cell border with multiple vesicles (Ves), granules, mitochondria and a coated pit. C) Detail of granule with multiple layers of membranes surrounded by mitochondria and ribosomes (Ri). Details of vesicular structures found in astrocytes in culture: D) Coated vesicle (CV) and uncoating vesicle (left CV) surrounded by ribosomes, intermediate filaments and mircrotubules (MT). E) Vesicle with ribosome and mitochondrium. F) Multivesicular body (MVB). G) Rough endoplamic reticulum (rER) with ribosomes. H) Golgi apparatus (Go). Astrocytes in culture with multiple cilia (Ci) next to nucleus (Nc; I). J) Detail of two cilia with microtubules and ribosomes. 85 3.1.2.3 Angiogenin Sorting after Internalisation

To investigate, which type of vesicle the angiogenin accumulates in or whether it is destined for degradation in lysosomes, rhAng-treated primary astrocytes were first labelled with the compartment specific, fluorescent dyes LysoTracker Red (LTR) and Monodansylcadaverine (MDC). LTR specifically detects acidic organelles such as lysosomes. It consists of a fluorophore coupled to a weak base, which easily permeates the cell. As the pH drops in acidic vesicles, the base gets protonated and the molecule is trapped within the compartment, probably bound to its membrane. Additionally, late lysosomes and autophagic vacuoles were labelled using the autofluorescent compound MDC, which is trapped inside these compartments by ion trapping and effective interaction with membrane lipids of autophagic vacuoles (Biederbick et al., 1995; Biederbick et al., 1999; Niemann et al., 2000). The experiment was performed by treating astrocytes with angiogenin for 1 h, 2 h, 6 h and 24 h, to be able to observe differences in labelling with time reflecting gradual degradation or sorting. Prior to fixation, lysosomes were labelled with 50 nM LTR for 30 min and 50 pM MDC for 10 min. Expectedly, confocal microscopy revealed good correlation of the two markers for acidic vesicles at all time points investigated (Figure 15; arrow heads). LTR staining was more restricted compared to MDC (Figure 15; comparison between middle two panels), which could reflect the difference in labelling specificity described above. By confocal microscopy, only little colocalisation of angiogenin staining with either of the two markers was detectable at the time points investigated (Figure 15; right panel). An& -. . . MDC

Figure 15: Some angiogenin containing vesicles colabel with lysosomal markers in primary astrocyte cultures. Primary cortical astrocyte cultures were treated with rhAng (1 pg/mL) for 1 h, 2 h, 6 h and 24 h or left untreated and costained with LysoTracker Red (LTR; 50 nM for 30 min) and monodansylcadaverine (MDC; 50 pM for 10 min; blue) prior to fixation and staining for angiogenin (green). Representative confocal images indicating colocalisation of angiogenin with both markers (bold arrows), neither of the two markers (fine arrows) or colocalisation of the two markers LTR and MDC (arrow heads). Depicted are representative images from two experiments performed with similar results. Scale bar 25 pm. Further experiments were performed to follow the vesicular route of angiogenin in primary astrocytes by immunocytochemically staining for the following marker proteins: The first marker tested was the small G-protein Rab 5, which is known to be involved in sorting of vesicles from different endocytic events (both clathrin- and caveolin-dependent) and labels early and sorting endosomes. Flotillin 1 has been described to appear on different non-classical endocytic intermediates. Rab 9 and Rab 1IA, two more small G-proteins, label late endosomes and recycling endosomes, respectively (reviewed by Doherty and McMahon, 2009). Primary astrocytes treated with rhAng for I h and stained for the endosomal markers were analysed by confocal microscopy. The experiment revealed that no marker tested did show overlap with angiogenin staining (Figure 16; arrow heads indicate angiogenin containing vesicles). Many vesicles staining positive for Rab 5 were observed evenly distributed throughout the cytoplasm (Figure 16A). Flotillin 1 labelling revealed less well-defined vesicular structures and strong accumulation of the protein at the cell surface (Figure 16B; bottom panel). Staining for Rab 9 showed many vesicles surrounding the nucleus (Figure 16C), whereas Rab 11A-staining vesicles were again less well-defined and found closer to the nucleus. Therefore, despite the antibodies displaying expected staining patterns, colabelling experiments left the exact identity of the angiogenin containing vesicles in primary astrocytes unknown. Ang Rab5 DAPl

4ng Flotillin 1 0 C -- DAPI

:. :. . . .(, , -,, . ,;b . a, 2; 3 . ', .. 4. ? . ' . . . irk . -4 . . r' .; Q 4ng Rab9 4ng Ra&t+ -. DAPI - DAPI

iabllA 3API Figure 16: No colocalisation of angiogenin containing vesicles with endosomal markers. Primary cortical astrocyte cultures were treated with rhAng (1 pglmL; red) for I h, fixed and costained for endosomal markers. A) Costaining for the early endosomal marker Rab 5 (green). B) Costaining for Flotillin 1 (green), which is found on non-clathrin and non-caveolin- associated vesicles. C) Costaining for the late endosomal marker Rab 9 (green). D) Costaining for the recycling endosomal marker Rab 11A (green). Arrow heads indicate angiogenin containing vesicles. Depicted are representative images from three experiments performed with similar results. Scale bars 25 pm. 3.1.3 Discussion

Initially, in primary mixed motoneuron cultures it was observed that only the non-neuronal cell population present within these cultures displayed punctate angiogenin staining following treatment with recombinant human angiogenin protein. The staining pattern was observed to be very similar in both astrocytes and microglia. No nuclear translocation of angiogenin in astrocyte cultures at various densities was seen, unlike shown before for endothelial cells and cancer cell lines (Moroianu and Riordan, 1994a; Tsuji et al., 2005). Therefore, it was decided to explore which other type of endocytic route could be involved in astrocytes. Transmission electron microscopy was performed to morphologically characterise the primary astrocytes used for this study especially with regard to vesicular structures present within these cells. As described previously (Eliasson et al., 1999), astrocytes abundantly express intermediate filaments in the cytoplasm and show thick microtubules throughout their cell bodies. The abundance in filaments was used to distinguish astrocytes from contaminating other cell types present in the primary cultures. Many different types of vesicles, both coated and uncoated, and vesicle-like structures were observed especially at the cell surface, pointing towards high endocytic activity within these cells in culture.

Additionally, astrocytes in culture displayed many electron dense granules with several layers of membranes. These could originate from endocytosed material after the initial preparation of the culture, as they strongly resemble the myelin sheath surrounding axons. Hardly any neurons survive under the culture conditions used, so it seems possible that astrocytes take up the remains of dead neurons and surrounding myelin sheath after tissue preparation. Another explanation for this observation could be autophagy. It has been observed that autophagosomes display very similar structural characteristics (Biederbick et al., 1995; Biederbick et al., 1999; Niemann et al., 2000) and autophagy is a process involved in cellular homeostasis, which is changed majorly by taking the cells in culture. It has been observed that primary astrocytes in culture contain some autophagosomes and upon exposure to gangliosides, which are constitutents of particularly neuronal cell membranes, autophagosome formation is increased (Hwang et al., 2010). In immunocytochemistry these granules showed up in all three channels routinely recorded even without labelled secondary antibodies, making automatic quantification of endocytic vesicles very difficult. Several attempts were made to analyse colocalisation based on confocal imaging data, which unfortunately failed due to the presence of these granules. However, it was easy to distinguish granules and vesicles for the experimentator based on subcellular localisation and triple staining, therefore it was chosen to perform manual counts of cells with angiogenin containing vesicles.

The initial experiment to characterise the vesicles in which angiogenin accumulates in primary astrocytes by colabelling with the two lysosomal markers LTR and MDC, revealed little overlap with angiogenin and no change in sorting even at very long time points of 24 h of treatment. Similarly, previous reports have shown that angiogenin uptake followed by nuclear translocation was independent of normal lysosomal function (Li et al., 1997). Colocalisation analysis was hampered by poor image quality of the MDC fluorescence, as the excitation (335 nm) and emission (508 nm) wavelengths are not included in the standard confocal microscope settings. Accordingly, settings were adjusted based on the DAPl spectrum, the most similar label commonly used with excitation at 360 nm and emission at 460 nm. The excitation wavelength was therefore not ideal to record MDC fluorescence, which resulted in high image background signal. Limited overlap of angiogenin staining with LTR and MDC thus suggested that only few of the vesicles observed were sorted along the classic endosome - late endosome - lysosome route.

Subsequently, immunocytochemical staining for endosomal markers was performed, however no colocalisation with angiogenin could be detected. Staining early, late and recycling endosomes with the markers Rab 5, Rab 9 and Rab 1IA, respectively (described above in Figure 16; pg. 89), displayed the expected vesicular pattern, eliminating possible doubts concerning antibody specificity. Similarly, staining for Flotillin 1 associated primarily with the plasma membrane and some vesicular structures within the cytoplasm as expected for a membrane-associated protein enriched in lipid rafts. As none of the endosomal markers tested showed much overlap with angiogenin staining, it was concluded that angiogenln accumulates in an as yet uncharacterised vesicular compartment, possibly for storage purposes or transcytosis, which may be characteristic for astrocytes,

As angiogenin containing vesicles could not be characterised in detail, the molecular mechanism of vesicle formation and budding was studied, hoping it might allow for conclusions to be drawn regarding the intracellular fate of angiogenln in astrocytes or the receptor mediating endocytosis. 3.2 Chapter lB

The Endocytic Mechanism of Angiogenin Uptake in Astrocytes

3.2.1 Introduction

3.2.1 .I Endocytic Mechanisms

Endocytosis is an essential process for the survival and homeostasis of cells and endocytic vesicles are continually formed at the plasma membrane of essentially all eukaryotic cells. The composition of the plasma membrane is controlled by endocytosis, thus it also determines cell interaction with and responses to the microenvironment. Depending on cell type and function many endocytic portals have been described (Figure 17), some of them highly specific like phagocytosis in macrophages, while others are commonly employed by most cell types like clathrin- or caveolin-dependent endocytosis (reviewed by Doherty and McMahon, 2009). Likewise, flotillins have been found to be involved in endocytic events in multiple different cell types, such as adipocytes, neurons and astrocytes (Stuermer et al., 2001 ; reviewed by Stuermer, 2011). Clathrin- and caveolin-independent endocytosis is cholesterol-dependent indicating the contribution of membrane microdomains. One such mechanism regulated by the small GTPase cdc42 is required for internalisation of some glycosylphosphatidylinositol (GPI)- anchored proteins into GPI-AP-enriched early endosomal compartments (CLICIGEEC), a process that can bypass Rab 5-positive endosomes. The small GTPase AI-F 6 has been implicated in a specific type of endocytic mechanism required for the uptake of CD59, MHC class I, mGluR7 and GPI- linked proteins. The internalisation of the interleukin 2 receptor P (IL2RP) occurs by a route distinct from any of the mentioned pathways. Large scale internalisation involving plasma membrane protrusions is called macropinocytosis, which is cholesterol- and actin-dependent (reviewed by Doherty and McMahon, 2009; and Stuermer, 201 1).

:aveolar-type ndocytosis Arf6-dependen endocytosis

- Dynamin 4 r Clathrin

Figure 17: Schematic representation of portals described in endocytic events. Electron microscopic images and fluorescence micrographs illustrate known structures in endocytosis. The best studied type of endocytosis is clathrin-mediated endocytosis (red). More specialised pathways are caveolar-type endocytosis (green), CLICIGEEC-type endocytosis of GPI- linked proteins (light blue), Arf6-dependent endocytosis (dark green), flotillin- dependent endocytosis (yellow) and the pathway described for interleukin-2- receptor p (IL2RP; pink). Cells of the immune system can internalise bacteria or large particles via phagocytosis (violet). Macropinocytosis allows for uptake of larger volumes of liquid (dark blue) and circular dorsal ruffles at the cell edges are also used for protein internalisation (light green). Entosis involves the uptake of a dying cell by a neighbouring cell (brown) (Doherty and McMahon, 2009). 3.2.1.2 Coated Pit Formation

Any type of endocytosis requires for the plasma membrane to engulf part of the extracellular medium, a process depending on the formation of endocytic cups by modifying membrane curvature. The best studied protein responsible for forming membrane pits is clathrin even though it is now known that many other proteins can similarly mediate pit formation without clathrin contribution. Clathrin was identified on 50 - 100 nm coated vesicles by electron microscopy (Pearse, 1976) and the molecular structure is well characterised: Clathrin forms three-limbed so-called triskelions consisting of one heavy and one light chain, which polymerise forming a polygonal lattice with intrinsic curvature. Clathrin-coated pits form about 2 % of the plasma membrane. These pits are then quickly internalised forming coated vesicles and the clathrin coat is shed shortly after endocytosis. Adaptor and accessory proteins trigger clathrin nucleation at the site of cargo internalisation, the two most important of the adaptor proteins being AP2 and AP180. Clathrin polymerisation alone is not sufficient for generating membrane curvature allowing for vesicle fission. Other proteins are involved that form more indirect scaffolds, cytoskeletal proteins and proteins that shape the membrane by directly inserting into the phospholipid bilayer (reviewed by Lodish, 2000; and Alberts, 2002; and Vassilieva and Nusrat, 2008; and Doherty and McMahon, 2009; and McMahon and Boucrot, 201 1).

3.2.1.3 Vesicle Budding

Formation of clathrin lattices at the plasma membrane prepares the coated vesicle for fission by bringing the membranes surrounding the neck into close proximity aided by additional proteins. The next crucial step in vesicle formation is membrane fission, which requires for the surface of the phospholipid bilayer to fuse via a hemifusion intermediate. This process is mediated by the GTPase dynamin in an energy-consuming reaction. Dynamin is recruited to the vesicle neck by curvature-sensing proteins and oligomerises by binding phosphatidylinositol 4,5-bisphosphate (PI 4,5BP) in the plasma membrane. While hydrolysing GTP, oligomerised dynamin pulls the membranes at the budding vesicle neck together and twists the membranes until fission occurs, a process that may involve actin to provide tension at the vesicle neck (Figure 18). Dynamin function has been observed to be necessary not only for clathrin-mediated endocytosis, but there is evidence for its involvement in caveolin-dependent endocytosis, as well as flotillin-mediated internalisation (reviewed by Lodish, 2000; and Doherty and McMahon, 2009).

Figure 18: Schematic representation of vesicle fission with the aid of the GTPase dynamin. Upon recruitment to the neck of a budding vesicle, dynamin oligomerises around the vesicle neck. GTP hydrolysis generates the force required to further constrict the neck to bring the membranes into close enough apposition for hemifusion to occur between two monolayers. A second step of hemifusion completes fission of the vesicle from the plasma membrane (Doherty and McMahon, 2009).

3.2.1.4 Studying the Endocytic Mechanism

Endocytic events can be studied by a variety of methods, the easiest being pharmacological inhibition of distinct endocytic pathways. Very early observations on polymorphonuclear leukocytes demonstrated the inhibition of receptor-mediated endocytosis in hyperosmolar medium (Daukas and Zigmond, 1985), still a very popular method to inhibit clathrin-mediated endocytosis (reviewed by Ivanov, 2008). It was later shown that clathrin coat formation was disrupted by increasing the osmolarity of the medium in 96 fibroblast cultures, as clathrin accumulates in small microcages instead of forming normal coated pits (Heuser and Anderson, 1989). However, hypertonic medium reduces both coated and uncoated pit formation (Carpentier et al., 1989), thus making this a very unspecific approach to study endocytosis (reviewed by Ivanov, 2008). Alternatively, the autofluorescent agent monodansylcadaverine (MDC) can be used to inhibit clathrin-mediated endocytosis, by inhibiting membrane-bound transglutaminase that is involved in endocytosis of many cargo receptor systems (Maxfield et al., 1979; Davies et al., 1980; Schutze et al., 1999; Gutierrez-Ortega et al., 2008). Another chemical commonly used to investigate endocytosis is the cyclic oligosaccharide methyl-P-cyclodextrin (MPCD), which extracts cholesterol molecules from the plasma membrane, thereby disrupting the integrity of lipid microdomains called lipid rafts. The water soluble molecule binds cholesterol within its ring structure by creating a hydrophobic environment surrounded by sugar groups at the molecule's core. This interferes with any endocytic process that requires cholesterol for vesicle formation, like caveolin- and flotillin-dependent endocytosis (Gutierrez-Ortega et al., 2008; Vercauteren et al., 2010) and some forms of clathrin-dependent internalisation (Rodal et al., 1999). As indicated above, results obtained with the mentioned inhibitors have to be interpreted with extreme care due many possible side effects of cell treatment and very specific responses of various cell types (reviewed by Ivanov, 2008; Vercauteren et al., 2010). Accordingly, to obtain more detailed information about endocytic mechanisms employed in a given model, other strategies have been developed. RNA interference to suppress the expression of components such as clathrin and AP2 has proven to be difficult as these proteins are very stably expressed. As a consequence, the more feasible approach to delineate endocytosis is to overexpress dominant-negative mutant proteins specifically involved in particular steps of protein internalisation. Mutant proteins derived from clathrin or the clathrin adaptor proteins AP180, Epsl5 or Epsin-I have been successfully used. Overexpression of the C-terminus of AP180, which still binds clathrin but cannot link clathrin to PI 4,5BP, specifically abolishes clathrin-dependent endocytosis for example (reviewed by Vassilieva and Nusrat, 2008; and Doherty and McMahon, 2009).

As described for the major endocytic portals, dynamin function can be inhibited by using inhibitory chemicals or overexpression of a dominant negative protein. Dynasore is an inhibitor of dynamin function identified in a screen of 16,000 small molecules (Macia et al., 2006). It specifically inhibits the GTPase activities of dynamin 1 and 2 and the mitochondria1 dynamin Drpl, which rapidly arrests pit formation at the stage of the almost fully formed pit with very narrow neck or at half-formed pits in cell culture (Macia et al., 2006; Tu et al., 201 0). Alternatively, the more slowly acting approach of overexpressing a dominant-negative mutant form of the dynamin protein can be chosen. Substituting Lysine 44 by Alanine in the first of three conserved nucleotide-binding elements reduces dynamin's guanine nucleotide-binding affinity and its hydrolytic activity towards GTP (Damke et al., 1994). This dominant-negative dynamin mutant competes with endogenously expressed wildtype dynamin (Marks et al., 2001) and is commonly used to study the contribution of dynamin to endocytic events (reviewed by Vassilieva and Nusrat, 2008; and Doherty and McMahon, 2009).

Based on the observations described above with regard to angiogenin internalisation by primary astrocytes, it was attempted to characterise the molecular steps involved in angiogenin vesicle formation at the plasma membrane. The well established techniques elucidated above were used in combination with immunofluorescent microscopy and Western blotting as read-out of treatment results.

3.2.2Results

3.2.2.1 Angiogenin Endocytosis is Clathrin-Dependent

As most of the angiogenin containing vesicles did not stain for any of the common markers tested, it was investigated which cellular mechanism of uptake was responsible for the formation of cytoplasmic vesicles in 98 astrocytes. To distinguish the main endocytic routes, clathrin-mediated endocytosis was inhibited by the inhibitor monodansylcadaverine (MDC; Gutierrez-Ortega et al., 2008) and lipid raft-dependent endocytosis by the cholesterol extraction agent methyl-P-cyclodextrin (MPCD; Gutierrez-Ortega et al., 2008; Vercauteren et al., 2010). Primary astrocytes were pretreated for 1 h with 0.2 mM MDC or 0.24 % MPCD before adding rhAng for 1 h. Inhibition of clathrin-dependent endocytosis via MDC almost completely abolished formation of intracellular vesicles (Figure 19A; middle panel), whereas inhibition of lipid raft-dependent endocytosis showed no effect on angiogenin uptake by astrocytes (Figure 19A; right panel). Quantification of uptake confirmed significant inhibition of angiogenin uptake by MDC, reducing the number of vesicle-containing cells counted manually from 60 % without MDC to 15 % with MDC pretreatment (Figure 19B). The cytoskeleton plays major part in endocytic events as means of transporting vesicles to their sorting stations. To investigate the involvement of cytoskeletal components in budding and transport of angiogenin containing vesicles in astrocytes, the cells were treated with the microtubule-stabilising agent paclitaxel or colchicine, which disrupts microtubules. Before rhAng treatment, primary astrocytes were incubated for Ih with paclitaxel (I pg/mL) or colchicine (4 pg/mL). Confocal imaging of treated cells revealed that both paclitaxel and colchicine were acting on the cytoskeleton at the conditions used for this experiment, i.e. paclitaxel lead to the formation of thicker microtubule bundles within the cells (Figure 19C; bottom left image), whereas colchicine reduced intact microtubule filaments visible in the cytoplasm of treated cells (Figure 19C; bottom right image). However, neither paclitaxel nor colchicine pretreatment of astrocytes inhibited angiogenin uptake (Figure 19C; arrow heads show angiogenin containing vesicles), indicating no involvement of microtubules in angiogenin vesicle formation and transport. al MDC MBCD

R Ang GFAP DAPI.- -- P Neurobasal No analoflenin

Neurobasal MDC MPCD d------ColchicineI Ang ~u~ulinDAPl Figure 19: Angiogenin endocytosis in primary astrocytes is clathrin- dependent but independent of the microtubule cytoskeleton. Primary cortical astrocyte cultures were pretreated for Ih with monodansylcadaverine (MDC; 0.2 mM) to inhibit clathrin-mediated endocytosis or methyl+ cyclodextrin (MPCD; 0.24 %) to inhibit lipid raft-dependent endocytosis prior to rhAng (1 pgImL) treatment. A) Representative epifluorescent images of treated primary astrocyte cultures stained for angiogenin (red) and GFAP (green). B) Quantification of astrocytes with angiogenin containing vesicles by manual counting of representative images ("p 5 0.05; mean ? SD; n = 3 separate experiments). C)Representative confocal images of primary cortical astrocyte cultures pretreated with the microtubule-stabilising agent paclitaxel (1 pglrnL) or the depolymerising agent colchicine (4 pglmL) for I h prior to treatment with rhAng (1 pglmL) for I h. Samples were costained for angiogenin (red) and tubulin (green). Experiment was performed in duplicate with similar results. Arrow heads indicate angiogenin containing vesicles. Scale bars 25 pm. In light of the significant inhibition of angiogenin uptake into astrocytes by MDC, costaining of angiogenin-treated primary astrocytes with an antibody against clathrin heavy chain was performed, a major constituent of the clathrin coat. Confocal imaging of clathrin staining showed many vesicular structures in astrocytes, especially along the cell borders (Figure 20A; right panel). Angiogenin-treated astrocytes revealed some extent of overlap between clathrin and angiogenin staining (Figure 20A; indicated by arrow heads in left panel), suggesting initial internalisation of angiogenin in a clathrin-dependent manner and later shedding of the clathrin coat as the vesicles were sorted within the cell.

To confirm this result more specifically, primary astrocytes were transfected with rAP180 GFP, which leads to the expression of a truncated, GFP-tagged form of the AP180 protein. Wild type AP180 serves as an adaptor for clathrin at the vesicular membrane and the truncated protein prevents assembly of the clathrin coat at the plasma membrane as it lacks its membrane binding domain (Kind gift of Prof McMahon; Doherty and McMahon, 2009). Analysing transfected cultures by confocal microscopy showed that the astrocytes expressing the construct did not take up angiogenin into cytoplasmic vesicles while surrounding untransfected cells did exhibit the usual punctate staining pattern (Figure 20B; indicated by arrow heads in left panel). To exclude possible effects of transfection and overexpression on cellular protein uptake mechanisms, astrocytes were transfected with eGFP but no change in the punctate, cytoplasmic angiogenin staining following transfection and treatment could be observed (Figure 20B, right panel). A B 1 h Ann no Ana Ih Ang

Ang Clathrin DAPl Ang DAPl rAP180

Figure 20: Angiogenin is internalised by primary astrocytes in clathrin- coated pits. A) Primary cortical astrocyte cultures were treated with rhAng (1 pg/mL) for I h prior to fixation and costained for angiogenin (red) and clathrin heavy chain (green). Arrow heads indicate colocalisation of angiogenin and clathrin in vesicles. Depicted are representative confocal images from three separate experiments performed with similar results. B) Transfection of primary cortical astrocyte cultures with the truncated, GFP- tagged rAP180 clathrin adaptor protein or control GFP vector. Transfected cells (24 h) were treated with rhAng (1 pglmL) for 1 h prior to fixation and staining for angiogenin (red). Arrow heads indicate angiogenin containing vesicles. Representative confocal images from three separate experiments performed with similar results. Scale bars 25 pm. 3.2.2.2 Angiogenin Vesicle Budding Requires Dynamin Function

Clathrin-mediated endocytosis usually occurs with the help of the small GTPase dynamin, which is responsible for the scission of the budding vesicle. To investigate the mechanism of budding of angiogenin containing pits in astrocytes, primary cultures were transfected with a GFP-tagged, dominant-negative form of dynamin 1, which bears a mutation in residue 44 changing Lysine to Alanine, thereby preventing the binding of the nucleotide and interfering with the function of the endogenous protein (Damke et al., 1994). Transfected astrocytes were treated with rhAng and analysed by confocal microscopy, which showed inhibition of angiogenin uptake only in astrocytes expressing the mutated protein, while untransfected surrounding cells still internalised angiogenin into vesicles (Figure 21A; indicated by arrows heads in top panel). Again, to exclude possible effects of transfection and overexpression on angiogenin uptake by astrocytes, the cultures were also transfected with eGFP, which did not influence astrocytic angiogenin uptake (Figure 21A, bottom panel).

To confirm the results obtained, the cell permeable small molecule inhibitor dynasore was used to inactivate the endogenous dynamin protein present in astrocytes (Macia et al., 2006; Tu et al., 2010). Primary astrocytes were pretreated with dynasore for 30 min at two different concentrations to prevent toxic effects and to determine the optimal concentration required for inhibition before adding rhAng. At the lower concentration of 40 pM dynasore, inhibition of angiogenin uptake into astrocytes was only minor (Figure 21 B; middle panel), while at 160 VM very little punctate angiogenin staining could be observed and angiogenin containing pits seemed to accumulate at the cell surface (Figure 21B; indicated by asterisks in top image of right panel). Astrocyte morphology as indicated by GFAP staining remained intact even at the higher dynasore concentration used (Figure 218; top panel). This result was quantified by counting representative images, confirming a reduction of angiogenin uptake by astrocytes with 40 pM dynasore by 28 % and 160 pM dynasore by 70 % (Figure 21 C). Ang dnDyn DAPl

I h Ang Dynasore Dynasore x

I I Dynasore Dynasore I 4ng G FAP DAPl

Figure 21: Angiogenin vesicle budding in primary astrocytes requires dynamin function. A) Primary cortical astrocyte cultures were transfected (24 h) with the GFP-tagged, dominant-negative small GTPase dynamin or control GFP vector and treated with rhAng (1 pg/mL) for 1 h. Cells were stained for angiogenin (red). Arrow heads indicate angiogenin containing vesicles. Depicted are representative confocal images from three separate experiments performed with similar results. B) Pretreatment of primary astrocyte cultures with the dynamin inhibitor dynasore for 30 min at two different concentrations (40 pM and 160 pM) prior to adding rhAng (1 pg/mL) for I h. Representative epifluorescent images of cells fixed and stained for angiogenin (red) and GFAP (green). Arrow heads indicate angiogenin containing vesicles and asterisks angiogenin containing pits at the cell surface. C)Quantification of astrocytes with angiogenin containing vesicles by manual counting of representative images (*p 5 0.05; mean + SD; n = 3 separate experiments). Scale bars 25 pm. 3.2.2.3 Angiogenin Endocytosis is Sodium- and Heparin-Dependent

Previous reports on endocytosis suggest a strong dependence of endocytic events on both the osmolarity of the medium and the sodium and calcium concentration (Daukas and Zigmond, 1985; Heuser and Anderson, 1989; Jiang and Chen, 2009). Furthermore, ligand binding to receptors depends on the salt concentration in the medium. High affinity receptors recognise their ligands even at high salt concentrations at which low affinity binding sites are inaccessible (Ron et al., 1993; Reich-Slotky et al., 1994; Marchese et al., 1998). Preparative experiments performed had shown that in Neurobasal medium, the medium commonly used to culture different types of neurons, astrocytes readily internalised rhAng, whereas in DMEM, which is used for many other immortalised cell lines, this effect was not observed. To test whether any of the factors mentioned above influenced uptake of angiogenin into astrocytes, either the sodium or calcium concentration of Neurobasal medium were increased to the corresponding values of DMEM (3000 mglL to 6400 mg1L NaCI; 200 mglL to 264 mglL CaCI2). Additionally, the osmolarity of Neurobasal was elevated from 235 mOsm to 335 mOsm using sucrose to match the value measured for DMEM (Brewer et al., 1993). The astrocyte cultures were pretreated with modified medium for 3 h before adding rhAng for 1 h. Epifluorescent imaging of treated astrocytes showed that the modified media had very different effects on angiogenin internalisation: Adjusting osmolarity or CaCI2 concentration did not affect angiogenin uptake (Figure 22A; indicated by arrow heads in middle two images), whereas raising the NaCl concentration of Neurobasal strongly reduced angiogenin endocytosis (Figure 22A; right image). Quantification of astrocytic uptake of angiogenin by counting representative epifluorescent images revealed a reduction in astrocytes with punctate angiogenin staining by about 40 % on increasing the extracellular NaCl concentration (Figure 22B). This result was additionally confirmed by Western Blotting of angiogenin-treated astrocyte lysates after preincubation with modified Neurobasal and hardly any angiogenin signal was detected with increased NaCl (Figure 22C). Ang GFAP DAPl B V) 2 120,

- NB Ang NB NaCl NB Suc NB CaCI,

NB NaCl NB Suc NB CaCI, NB rh Ang Ang - + - + - + - + long 1 . - . - 1

Figure 22: Angiogenin internalisation by primary astrocytes depends on the extracellular sodium chloride concentration. A) Primary cortical astrocytes were pretreated for 3 h in Neurobasal (NB) adjusted to DMEM values for NaCI, osmolarity (with sucrose; Suc) or CaCI2 (3000 mg/L to 6400 mg/L NaCI; 235 mOsm to 335 mOsm; 200 mglL to 264 mg/L CaCI2). Following treatment with rhAng (1 pg1mL) for 1 h, cells were fixed and stained for angiogenin (red) and GFAP (green). Depicted are representative epifluorescent images. Arrow heads indicate angiogenin containing vesicles. Scale bar 25 pm. B) Quantification of astrocytes with angiogenin containing vesicles by manual counting of representative images (*p 5 0.05; mean k SD; n = 3 separate experiments). C)Western blot probed for angiogenin of astrocyte lysate treated under the same conditions. Experiment was performed in duplicate with similar results. The strong dependence of angiogenin uptake into astrocytes on extracellular sodium concentration suggested the involvement of heparansulfate proteoglycans in angiogenin endocytosis, as has been shown for bFGF (reviewed by Belting, 2003; and Dreyfuss et al., 2009). Therefore, it was tested whether adding heparin to the medium or heparinase I preincubation of the astrocytes resulted in any changes in the observed punctate angiogenin staining. It has been shown that angiogenin binds heparin (Soncin et al., 1997), therefore extracellular heparin may compete with the receptor. Heparinase I is a bacterial enzyme which removes heparansulfates from the cell surface thereby impeding heparansulfate proteoglycan-aided endocytic events (Roghani and Moscatelli, 1992). Primary astrocytes were pretreated with 10 pglmL heparin or 2.5 UlmL heparinase I for 2 h before adding rhAng. lmmunocytochemistry showed that heparin in the medium completely abolished any angiogenin uptake (Figure 23A; left panel), while the effect of heparinase I was less pronounced (Figure 23A; middle panel). Manual counts were performed to quantify angiogenin endocytosis from representative images, which confirmed almost complete inhibition of angiogenin endocytosis by heparin to less than 5 %, but the reduction of uptake by heparinase I preincubation by 28 % was too small to test significant (Figure 23B). Western Blotting of astrocyte lysates treated with heparin and angiogenin or preincubation with heparinase I showed that very little angiogenin was detected with heparin treatment (Figure 23C), supporting the involvement of heparansulfate proteoglycans in angiogenin endocytosis by astrocytes. Heparinase I Neurobasal

DAPI 6 %Ck -4b., t)! ' 4

Ang GFAP DAPl

Heparin Heparinase I Neurobasal rh Ang Ann + - + - + - long

0. - Heparin Heparin- Neuro- nase l basal

Figure 23: Heparin inhibits internalisation of angiogenin by primary astrocytes. A) Primary cortical astrocytes were pretreated for 2 h with heparin (10 pglmL) or heparinase 1 (2.5 UImL) prior to treatment with rhAng (1 pglmL) for 1 h. Depicted are representative images of cells fixed and stained for angiogenin (red) and GFAP (green). Arrow heads indicate angiogenin containing vesicles. Scale bar 25 pm. B) Quantification of astrocytes with angiogenin containing vesicles by manual counting of representative images (*p 5 0.05; mean + SD; n = 3 separate experiments). C) Western blot probed for angiogenin of astrocyte lysate treated under the same conditions. Experiment was performed in duplicate with similar results. 3.2.3 Discussion

Further studies to delineate angiogenin internalisation by primary astrocytes in culture were performed with the aid of inhibitors of clathrin-dependent and lipid raft-dependent endocytosis. Using inhibitors on cell cultures is always difficult, as the inhibitors are never truly specific for any individual process, but do have other effects on cells that can interfere with their general homeostasis (reviewed by Ivanov, 2008). It is thus very important to optimise treatment protocols with regard to inhibitor concentration and incubation times to avoid toxic effects and to work at the most effective conditions possible. Primary astrocytes treated with the two inhibitors MDC and MPCD did not display gross morphological changes (Figure 19A; pg. 100) indicating their viability, however MDC strongly inhibited angiogenin internalisation, clearly pointing towards a clathrin-dependent uptake mechanism.

In accordance with previous studies, it was observed that angiogenin uptake was independent of microtubules (Li et al., 1997). Neither stabilising nor depolymerising the microtubule network had any effects on vesicle formation, even though the microtubule network was evidently affected as shown by immunofluorescent staining for tubulin (Figure 19C; pg. 100). This strongly indicates the involvement of other elements of the cytoskeleton, like actin or intermediate filaments, in internalisation and routing of angiogenin in primary astrocytes.

Due to the limitations drawing conclusions solely based on inhibitor studies, it was chosen to use immunocytochemistry to observe colocalisation of angiogenin containing vesicles with clathrin heavy chain, a major component of the clathrin coat (reviewed by Mousavi et al., 2004). Partial colocalisation of angiogenin and clathrin in vesicular structures was seen, supporting the hypothesis that angiogenin is internalised by a clathrin-dependent mechanism. The observation that the two proteins colocalise only partially indicates that the clathrin coat is shed with further sorting of angiogenin in primary astrocytes. This was confirmed more specifically by transfecting a truncated, GFP-tagged form of the clathrin adaptor protein AP180, which

109 does not contain the membrane-binding domain of the native protein but associates normally with clathrin (reviewed by Doherty and McMahon, 2009). Overexpression of rAP180 in primary astrocytes clearly inhibited angiogenin endocytosis further supporting the view that the endocytic mechanism involved in this model system involves classic clathrin-mediated endocytosis.

The next important step in vesicle formation is the process of membrane fission, which requires the use of energy to separate the membranes. The most common and best studied protein responsible for membrane fission in various endocytic pathways including clathrin-dependent endocytosis is dynamin, thus angiogenin uptake in primary astrocytes overexpressing the dominant-negative GFP-tagged K44A (Damke et al., 1994) was investigated and shown to be inhibited. Furthermore, dynamin function was inhibited with the small molecule inhibitor dynasore, which strongly reduced angiogenin endocytosis. As endocytosis inhibitors can have toxic effects on cells, the experiment was performed at two different concentrations, however no gross morphological changes were observed (Figure 21B; pg. 104). Dynasore selectively inhibits the small GTPases dynamin 1, dynamin 2 and Drpl (mitochondria1 dynamin) within seconds, leading to the accumulation of coated pit intermediates (Macia et al., 2006). Similar effects were observed in astrocyte cultures treated with dynasore, thus efficient blocking of dynamin function was confirmed, however binding of angiogenin to its receptor was not altered. Thus, dynamin is responsible for fission of angiogenin containing vesicles in astrocytes.

The initial observation that angiogenin internalisation was visible as punctate staining in primary astrocytes in coculture with motoneurons in Neurobasal medium was made very difficult to interpret at first, as no vesicles were observed in full DMEM following angiogenin treatment. Differences in media formulation were investigated and the two major differences between the two media are osmolarity and sodium chloride concentration, both of which strongly influence endocytic events by affecting coat formation and receptor binding (Daukas and Zigmond, 1985; Heuser and Anderson, 1989; Ron et al., 1993; Reich-Slotky et al., 1994; Marchese et al., 1998). The experiment showed that increasing the sodium chloride concentration in Neurobasal almost completely abolished angiogenin endocytosis. Neurobasal is a medium commonly used to grow neuronal cells and was originally developed to optimise the growth and survivial of rat hippocampal embryonic neurons (Brewer et al., 1993), whereas the formulation of DMEM is based on observations made using mouse fibroblasts and the human carcinoma cell line HeLa (Eagle, 1955). This difference in behaviour of astrocytes is quite striking, as one would assume the microenvironmental conditions in the central nervous system would allow for optimal neuron survival. Physiological sodium concentration in the CSF is very similar to plasma at 145 - 155 mM (Harrington et al., 2010), but local ion concentrations in the brain tissue are strongly dependent on local nerve activity (reviewed by Somjen, 2002), thus conditions surrounding astrocytes vary.

Furthermore, this observation quite strongly indicated that receptor binding was inhibited in DMEM at higher sodium chloride concentration, supported by the lack of cell surface staining of cells with the angiogenin antibody (Figure 22A; pg. 106; left image). Binding of growth factors to low affinity receptors has been shown to depend on extracellular sodium concentration (Ron et al., 1993; Reich-Slotky et al., 1994; Marchese et al., 1998). This effect is caused by the strongly anionic glycosaminoglycan chains, which interact with sodium ions present in the extracellular space thus reducing charge density (Ahl et al., 2009). Accordingly, binding of a ligand releases the counterion, a process that reaches equilibrium depending on the concentrations of sodium ions and ligand molecules present. Heparansulfate proteoglycans serve as coreceptors for bFGF for example (Deguchi et al., 2002; Berry et al., 2003; Zhang et al., 2003; Tkachenko et al., 2004) and as angiogenin had been shown to bind heparin (Soncin et al., 1997), it was speculated that heparansulfates are involved in endocytosis of angiogenin by astrocytes, too.

Adding heparin to the culture medium to compete for angiogenin binding completely abolished angiogenin endocytosis, while pretreatment with the bacterial enzyme heparinase I only slightly reduced angiogenin uptake. This result clearly demonstrates that heparin competes with the receptor on the cell sutface for angiogenin binding at the concentration used. Angiogenin is a basic protein displaying multiple positively charged residues at the molecule's surface, which could account for the observed binding to heparin. However, mutating these residues has been shown not to majorly influence heparin binding, thus indicating that the interaction of angiogenin with heparin is glycosaminoglycan specific (Soncin et al., 1997). The observation that heparinase I pretreatment was less efficient can therefore be interpreted as follows: Heparinase enzymes show different substrate specificities (Nader et al., 1999) and previous reports show that heparinase treatment effects can be variable depending on the heparansulfate proteoglycan target (Lin et al., 2005). Thus, the enzyme may not have removed the specific glycosaminoglycan residues required for angiogenin recognition by its heparansulfate receptor.

It was concluded that angiogenin is internalised by astrocytes via clathrin- dependent endocytosis requiring functional dynamin. The receptor initially recognising angiogenin on astrocytes was speculated to be a heparansulfate proteoglycan. 3.3 Chapter IC

Syndecan 4 is the Receptor for Angiogenin on Astrocytes

3.3.1 Introduction

3.3.1 .I Cell Surface Proteoglycans

Following the observations described in the previous chapter, that heparin inhibited angiogenin endocytosis by primary astrocytes, along with previous reports of angiogenin binding heparin and heparansulfate proteoglycans (Soncin et al., 1994; Soncin et al., 1997), it was concluded that heparansulfate proteoglycans were the best receptor candidates for angiogenin uptake by astrocytes.

Proteoglycans are synthesised by almost every cell type in metazoan organisms and consist of a core protein attached to glycosaminoglycan sugar chains. Proteoglycans contribute majorly to the composition of the ECM, while some of them are secreted and others remain attached at the cell surface. Besides attaching to fibrous collagens and fibronectin, they bind a wide variety of ligands, such as bFGF and VEGF, via basic amino acid stretches displayed on their surface, as well as other types of secreted proteins such as proteases. Therefore, proteoglycans are required to anchor cells to the ECM. Additionally, this allows for proteoglycans to regulate communication between cells by selectively sequestering proteins to either activate or inhibit their molecular function or to influence receptor signalling triggered by ligand binding (reviewed by Lodish, 2000; and Alberts, 2002; and Belting, 2003).

Two major types of cell surface proteoglycans are commonly distinguished based on their structural characteristics: The glypicans, which insert into the plasma membrane via a GPI anchor, are critically involved in development and morphogenesis. The most common and best studied type of cell surface proteoglycan are the syndecans. The core protein of syndecans spans the plasma membrane, so they have a short cytosolic domain and a long extracellular domain with at least three heparansulfate chains attached at the very N-terminal end of the protein. There are four different types of syndecans with very similar overall structure: Syndecans 1 to 4 (reviewed by Lodish, 2000; and Belting, 2003; and Multhaupt et al., 2009). The carbohydrate is separated from the plasma membrane by a domain responsible for integrin binding (Figure 24). The transmembrane domains of all syndecans are very similar in amino acid sequence and are responsible for syndecan homodimer or oligomer formation. The cytoplasmic domain can be subdivided into three distinct regions: A variable region (V), unique to each syndecan type, separates the two highly conserved domains (C1 and C2), which are characteristic for this class of proteins. The transmembrane and cytoplasmic domains can trigger signalling across the plasma membrane, however they do not display any intrinsic catalytic activity (reviewed by Zimmermann and David, 1999; and Tkachenko et al., 2005; and Lambaerts et al., 2009; and Multhaupt et al., 2009). Heparan sulphate bearing domain; HS chains interact with growth factors and fibronectin.

Cell-binding domain; mediates pl integrin dependant mesenchymal cell adhesion through a conserved NXlP motif.

- Transmembrane domain; which drives syndecan dimer formation. b...... a*-+..... - - Cytoplasmic domain; Cytosol contains three sub- B 3--domains C1 ,V and C2. Figure 24: Schematic overview of syndecan structure. Syndecans consist of a core protein, which is attached to gycosaminoglycan chains at the membrane distal extracellular region. The membrane proximal domain binds integrins and therefore mediates cell adhesion. The transmembrane domain, as well as parts of the intracellular region, is responsible for syndecan dimerisation. Syndecan proteins contain two conserved cytoplasmic domains and one variable region, which is unique to each syndecan (Multhaupt et al., 2009).

3.3.1.2 Syndecan 4

The best-characterised of the syndecans with regard to signalling is syndecan 4 (Syn4). Many different interaction partners of syndecan 4 at its cytoplasmic tail have been identified that contribute to signalling. Binding partners at the C1 domain of syndecan 3, which is identical to syndecan 4, are cortactin, P-tubulin and the two tyrosine kinases c-src and Fyn. These interactions indicate tight association of syndecans with the cytoskeleton. Syndesmos specifically binds the syndecan 4 Cl domain. The C2 domain contains two PDZ-binding motifs, and binds syntenin, CASK and synectin. The variable domain binds PI 4,5BP, an interaction that may be required for the formation of higher order oligomers of syndecan 4. Additionally, the variable region is also responsible for syndecan 4 binding to the plasma membrane-associated protein kinase C alpha (PKCa). Oligomerisation of 115 syndecan 4 and interaction with PI 4,5BP are required for this mode of PKCa activation (Oh et at., 199713, a; Oh et al., 1998; Keum et al., 2004). Classically, PKCa was thought to be activated by calcium, phosphatidylserine and diacylglycerol, a cleavage product derived from PI 4,5BP (reviewed by Zimmermann and David, 1999; and Lodish, 2000; and Simons and Horowitz, 2001; and Alberts, 2002; and Multhaupt et al., 2009). Syndecan 4 is associated with the formation of stress fibers and focal adhesions, sites of cell attachment to the substratum, involving both PKCa and the small GTPase Rho, which activates focal adhesions kinase (FAK; reviewed by Bass and Humphries, 2002; and Wilcox-Adelman et al., 2002b; and Lambaerts et al., 2009).

As mentioned before, proteoglycans can bind and sequester extracellular growth factors. Syndecan 4 has also been shown to interact with receptor tyrosine kinases and modulate signalling following ligand binding to the receptor. Most studies have been performed on syndecan 4 contribution to bFGF signalling and internalisation (reviewed by Bass and Humphries, 2002; and Tkachenko et al., 2005; and Murakami et al., 2008; and Lambaerts et al., 2009). The endocytic mechanism of bFGF uptake by endothelial cells is activated by syndecan 4 clustering in lipid rafts, but does not require clathrin or dynamin (Tkachenko and Simons, 2002; Tkachenko et al., 2004). However, internalisation of bFGF by endothelial cells derived from the brain has been shown to be dependent on the secreted proteoglycan perlecan (Deguchi et at., 2002), highlighting the variability in endocytic routes employed by cargo proteins.

Syndecan 4 shows almost ubiquitous expression throughout the body, whereas the other three syndecan proteins show more specific expression patterns. Interestingly, it has been observed that syndecan 4 in the adult rat brain is expressed by the glial cell population, while syndecans 2 and 3 are found on neurons (Hsueh et al., 1998; Ethell and Yamaguchi, 1999; Hsueh and Sheng, 1999; lseki et al., 2002). Syndecans are implicated in a variety of diseases such as cancers and infections and contribute to wound healing and angiogenesis as well as brain repair (reviewed by Properzi et al., 2003; and Fears and Woods, 2006). Studies on injured mouse brains revealed upregulation of all four types of syndecans in reactive astrocytes (Iseki et al., 2002), however there are no reports on the contribution of syndecans to ALS.

3.3.1.3 Colocalisation Analysis

In light of the function of the cell surface proteoglycan syndecan 4 detailed above, and on the basis of earlier observations made during this study, it was chosen to investigate a possible contribution of syndecan 4 to angiogenin internalisation by primary astrocytes in culture. Techniques commonly used to study protein-protein interactions such as immunofluorescent colocalisation and coimmunoprecipitation were employed, supported by a new strategy called Proximity Ligation Assay (PLA; explained on pg.59). This method in situ detects two epitopes in close proximity, as recognised by primary antibodies, which may be located on the same or different proteins. The assay can be performed on tissue samples without the requirement of protein overexpression for signal detection, as rolling circle DNA amplification greatly enhances signal intensity from any individual site of interaction. Furthermore, it allows for the exact localisation of the points of interaction within the cell investigated.

3.3.2 Results

3.3.2.1 Angiogenin Binds Syndecan 4 on Astrocytes

Based on the previously detailed observation that angiogenin internalisation by astrocytes is abolished by adding heparin to the medium and by increasing the sodium chloride concentration, heparansulfate proteoglycans were speculated to serve as angiogenin receptors. Previous studies have shown that the proteoglycan syndecan 4 mediates bFGF uptake (Tkachenko and Simons, 2002; Tkachenko et al., 2004). Therefore, it was investigated whether angiogenin internalisation by astrocytes may be mediated by syndecan 4. Angiogenin-treated primary astrocytes were immunocyto-

117 chemically stained for angiogenin and syndecan 4. Confocal microscopy of primary astrocyte cultures confirmed basal expression of syndecan 4 (Figure 25A; bottom panel), as has been shown previously (Hsueh and Sheng, 1999). In rhAng-treated astrocytes colocalisation of angiogenin and syndecan 4 in vesicles was observed (Figure 25A; indicated by arrow heads in top panel).

Protein colocalisation in fluorescent microscopy not necessarily reflects direct interaction of the two proteins stained for. To obtain further insights into the interaction of the two proteins angiogenin and syndecan4, coimmunoprecipitation studies were performed, as this would allow for detecting proteins present within a complex by direct or indirect interaction. Precipitation of angiogenin from astrocyte lysates was performed with the same polyclonal goat antibody as was used for all immunofluorescent staining experiments for angiogenin. A polyclonal goat anti-Rbx2 antibody served as negative control to exclude unspecific binding to the goat IgG antibody. To increase receptor binding specificity but to prevent complete internalisation and partial degradation, two different treatment protocols with angiogenin were used: The first one allowed for 30 minutes of internalisation at 37 "C followed by two hours of incubation at 4 "C to increase binding specificity. The second one was performed without the internalisation period at 37 "C. The lysis buffer for precipitation was adjusted to the same NaCl concentration as Neurobasal to ensure binding of angiogenin to its receptor was not disrupted due to a change in milieu upon cell collection and lysis. As a positive control for syndecan 4 expression, astrocyte lysate in RlPA buffer was loaded. Both treatment protocols lead to very similar results (Figure 25B; 2"d and 4th lanes). Very low levels of angiogenin were detected in the Rbx2 precipitate compared to the angiogenin precipitate (Figure 25B; 8th and 1oth lane). However, when comparing the precipitate obtained with the angiogenin and the Rbx2 antibodies probed for syndecan 4, bands are also detected at the correct level in the Rbx2 precipitate. The angiogenin enrichment by precipitation with treatment was 12 fold in this experiment, whereas the enrichment of syndecan 4 was only 1.5 fold. DAPl Q@,

An!- I DAPl

at" a Anaioaenin- - at- a Rbx2 Astro- rh Ana- 37"C0/4"C 4°C no Ang 37"C/4"C 4°C no Ang cytes lys prec lys prec lys prec lys prec lys prec lys prec RIPA long m; -,-.

Figure 25: Angiogenin and syndecan 4 colocalise in primary astrocytes. A) Representative confocal images of primary cortical astrocyte cultures treated with rhAng (1 pg/mL) for Ih prior to fixation and stained for angiogenin (red) and the heparansulfate proteoglycan syndecan 4 (Syn4; green). Arrow heads indicate colocalisation of angiogenin and syndecan 4 in vesicles. Experiment was performed in triplicate with similar results. Scale bar 25 pm. B) Coimmunoprecipitation of angiogenin with syndecan 4 from primary cortical astrocyte cultures. Cells were treated with rhAng (1 pglmL) for 30 min at 37 "C followed by 2 h at 4 "C or just 2 h at 4 "C and angiogenin was precipitated using a goat anti-angiogenin antibody and protein AIG agarose beads. To control for unspecific binding to the goat antibody, precipitation was also performed with a goat anti-Rbx2 antibody. Collected lysate (lys) and precipitate (prec) were loaded. The experiment was performed in quadruplicate. As coimmunoprecipitation did not lead to satisfactory results, the in situ Proximity Ligation Assay (PLA) was performed (Soderberg et al., 2006), which detects interactions of proteins in fixed cells and therefore gives more information about the exact localisation of the interaction partners compared to immunoprecipitation. Instead of using fluorescently-labelled secondary antibodies, the primary antibodies were detected by antibodies attached to DNA oligonucleotides. Provided these are in close enough proximity to be linked by a connector oligonucleotide, they serve as a template for rolling circle amplification. This greatly amplifies the signal and the DNA can be detected with a fluorescently-labelled DNA probe. At every protein interaction site (c 40 nm) a single red spot is generated. Primary astrocytes were seeded onto glass bottom 8-well slides and angiogenin treatment was performed after 48 h. Fixed astrocytes were subjected to the proximity ligation procedure followed by staining with phalloidin and DAPl to distinguish individual cells. Samples were analysed by both confocal and epifluorescent imaging, which revealed many points of interaction of syndecan 4 and angiogenin in rhAng-treated astrocytes (Figure 26A; top left images), much fewer spots in untreated astrocytes (Figure 26A; bottom left image) and hardly any signal without primary antibodies (Figure 26A; bottom middle image). There was little background by unspecific antibody interaction as shown by using an antibody against GFAP in combination with the angiogenin antibody (Figure 26A; right panel). For quantification of the spots, the Blob Finder software provided by Olink was used on the epifluorescent images. In angiogenin-treated cells 18 points of interaction were detected on average compared to only 2 spots in untreated astrocytes (Figure 268). This result strongly supported a syndecan 4-angiogenin interaction in astrocytes.

To learn more about the site of interaction between syndecan 4 and angiogenin within the treated astrocytes, the cells were first transfected using tGFP-tagged syndecan 4 followed by treatment with angiogenin after 24 h and successive PLA analysis. Confocal images of the transfected astrocytes showed accumulation of PLA spots within vesicles containing tGFP-tagged syndecan 4 (Figure 26C), which further underscored the previous findings. A aAng aSyn4 aGFAP aAng

aAng aSyn4 no primary antibody aGFAP aAng PLA Phalloidin DAPl

Ang no Ang no Ang

LA Syr14uFP DAPl

Figure 26: Angiogenin and syndecan 4 are found in close proximity in primary astrocytes. A) Proximity ligation assay (PLA) on primary cortical astrocyte cultures treated with rhAng (1 pg/mL) for 1 h. Cells were stained with primary antibodies against angiogenin (aAng) and syndecan 4 (aSyn4). The following controls were included: no primary antibodies or mouse anti- angiogenin in combination with rabbit anti-GFAP (aGFAP). Phalloidin staining was performed to visualise cell borders (green), red spots represent points of interaction between the two primary antibodies detected by PLA. Representative confocal images of treated and control conditions shown. B) The blob finder software provided by Olink was used to quantifiy PLA spots detected within individual cells from epifluorescent images (*p I 0.05; mean + SD; n > 350 cells in three independent experiments). C) Representative confocal images of PLA on tGFP-tagged syndecan 4 transfected (Syn4 GFP; 24 h) primary cortical astrocyte cultures treated with rhAng (1 vg/mL) for 1 h. Arrow heads indicate PLA signals in syndecan 4 containing vesicles. Experiment was performed in duplicate with similar results. Scale bars 25 pm. 121 3.3.2.2 Suppressing Syndecan 4 Expression Inhibits Angiogenin Uptake

To prove that syndecan 4 is essential for angiogenin internalisation by astrocytes, shRNA technology was used to suppress syndecan 4 expression. Four commercially available GFP-coexpressing shRNA expression plasmids were tested for syndecan 4 gene silencing in astrocyte cultures by transient transfection. Due to limited transfection efficiency in primary astrocyte cultures, it was impossible to quantify the inhibition of syndecan 4 expression by Western blotting. Instead, confocal microscopy was chosen to analyse costaining of GFP-expressing cells with syndecan 4 immunofluorescence (Figure 27A). The two most effective expression vectors (designated shRNAl and shRNA2) were chosen for analysis of the effects of syndecan 4 gene silencing on angiogenin uptake by astrocytes.

After transfection, cells increasingly detached from coated coverslips, making it necessary to perform rhAng treatment after 48 h to avoid losing cells for later analysis. Cells transfected with the specific shRNA clones in primary astrocyte cultures displayed reduced angiogenin uptake by fluorescence microscopy, while surrounding untransfected cells internalised angiogenin normally (Figure 27B; arrow heads in top two panels). Transfection of the scrambled DNA sequence did not lead to the same effect of reducing angiogenin endocytosis (Figure 27B; bottom panel). Punctate internalisation of angiogenin in these cell cultures was observed in 76 % of cells. Analysis of GFP-expressing cells showed that transfection with the specific shRNAl or shRNA2 reduced angiogenin uptake by 56 % and 48 %, while scrambled shRNA-transfected controls reduced uptake by only 17 % (Figure 27C). 4

I i d Ana GFP '.-,a -1 '--,-- '--,-- Ang GFP DAPl a I * 1 c I,, -

shRNAl shRNA2 Scramble Figure 27: Angiogenin internalisation by primary astrocytes can be inhibited by suppressing syndecan 4 expression. A) Primary cortical astrocyte cultures were transfected with two different GFP syndecan 4 shRNA clones or a scrambled sequence and costained for syndecan 4 (Syn4; red) 48 h after transfection. Depicted are representative confocal images from three experiments performed with similar results. B) Representative pseudoconfocal images of GFP syndecan 4 shRNA- transfected (48 h) primary astrocyte cultures treated with rhAng (1 pg/mL) for 1 h prior to fixation and staining for angiogenin (red). Arrow heads indicate angiogenin containing vesicles. C) Quantification of GFP-positive cells with angiogenin containing vesicles by manual counting of representative images. Angiogenin uptake was observed in 76 % of cells in these cultures (*p I0.05; mean + SD; n < 30 cells in three independent experiments). Scale bars 25 pm. 123 3.3.2.3 Syndecan 4 Can Induce Uptake of Angiogenin

It has previously been published that angiogenin internalisation into endothelial cells requires the presence of a putative 170 kDa receptor (Hu et al., 1997). However, to date this receptor is has not been characterised. To exclude possible contribution of this receptor to angiogenin endocytosis by astrocytes, NSC34 cells were transfected with tGFP-tagged syndecan 4. This is a hybrid cell line from embryonic mouse spinal cord and mouse neuroblastoma cells with characteristics of primary motoneurons (Cashman et al., 1992). These cells were chosen as they did not normally internalise angiogenin (observations made in preparation of the experiment) nor did they basally express syndecan 4 in accordance with previous reports on neurons in the adult rat brain (Hsueh and Sheng, 1999). Transfection of NSC34 cells with tGFP syndecan 4 prior to treatment with angiogenin induced endocytosis of angiogenin into these cells resulting in punctate staining like observed in astrocytes (Figure 28A; arrow heads in top two panels). Costaining of the transfected NSC34 cells with an antibody against syndecan 4 confirmed an overlapping staining pattern of the transfected, overexpressed protein and the protein labelled by the antibody (Figure 28B). However, no quantification of angiogenin containing vesicles was possible due cell detachment during transfection and immunostaining, leading to cell clusters in which individual cells could hardly be distinguished. Nevertheless, to exclude possible effects of transfection and overexpression on cellular protein uptake mechanisms, NSC34 cells were transfected with eGFP but no punctate, cytoplasmic angiogenin staining following treatment was observed (Figure 28C). - . C? ,,;! ~n~ Syn4 GFP DAPl -- -I Ang Syn4.- GFP DAPl 4 Angiogenin containing vesicles

<-', , I ., , ., -.

1 .. , '* ', q , ..,.., . . ' ' . :,4' svn4 Syn4 GFP DAP/ (-. , Syn4 Syn4 GFP DAPl

Ang eGFP DAPl

Figure 28: Syndecan 4 expression is sufficient to induce angiogenin internalisation in NSC34 cells. A) Motoneuron-like NSC34 cells were transfected (24 h) with tGFP-tagged syndecan 4 (Syn4 GFP) prior to treatment with rhAng (1 pglmL) for I h. Fixed cells were stained for angiogenin (red). Arrow heads indicate angiogenin containing vesicles. Depicted are representative confocal images from two separate experiments performed with similar results. B) NSC34 cells transfected (24 h) with tGFP- tagged syndecan 4 prior to fixation and staining for syndecan 4 (red). Depicted are representative confocal images from two separate experiments performed with similar results. C) Control GFP vector-transfected NSC34 cells treated with rhAng (1 pg1mL) for 1 h prior to fixation and staining for angiogenin (red). Depicted are representative confocal images from two separate experiments performed with similar results. Scale bars 25 pm. 125 3.3.2.4 Signalling Elicited by Angiogenin Binding Syndecan 4

The heparansulfate proteoglycan syndecan 4 is not only involved in endocytosis of bFGF (Tkachenko and Simons, 2002; Tkachenko et al., 2004), but it also modulates bFGF signalling in vivo (Berry et al., 2003; Zhang et al., 2003). Syndecan 4 has been shown to be able to activate several different signalling cascades (reviewed by Tkachenko et al., 2005; and Multhaupt et al., 2009), but two of the most commonly activated pathways involve PKCa and focal adhesions kinase (FAK; Oh et al., 1997a; Oh et al., 1997b; Wilcox-Adelman et al., 2002a; Keum et al., 2004). Primary astrocyte cultures treated with rhAng for I h were immunocytochemically stained for FAK or PKC and samples were analysed by confocal microscopy. The experiment did not reveal any colocalisation of angiogenin staining and FAK (Figure 29A), while the staining pattern showed most FAK protein was found in foci mainly along the attachment surface of the cells, most likely corresponding to focal adhesions. Costaining for angiogenin and PKC revealed some points of colocalisation at vesicles (Figure 29B), suggesting PKC may be involved in angiogenin signalling in astrocytes.

All efforts to further delineate intracellular signalling in astrocytes in response to angiogenin by Western blotting, ca2+ imaging and CAMP measurements were unsuccessful. t I . ... , ... .

.. 4 -... ,- * I .. ..*- .. F

C, '4 >, ...

a..; . "b ' .1. 8

. ->..,- PDAPI FAK r' .I... Ang FAK DAPl

2 DAPl

Figure 29: Syndecan4 signalling in response to angiogenin internalisation by primary astrocytes most likely involves PKC. A) Primary cortical astrocyte cultures were treated with rhAng (1 pg/mL) for 1 h prior to fixation and stained for anigiogenin (red) and focal adhesions kinase (FAK; green). Depicted are representative confocal images of two separate experiments performed with similar results. B) Primary cortical astrocyte cultures were treated with rhAng (1 pglmL) for 1 h prior to fixation and stained for anigiogenin (red) and protein kinase C (PKC; green). Arrow heads indicate colocalisation of angiogenin and PKC in vesicles. Depicted are representative confocal images of two separate experiments performed with similar results. Scale bars 25 pm. 3.3.3 Discussion

Literature research on heparansulfate proteoglycans which could be involved in angiogenin endocytosis, pointed towards involvement of syndecan 4 in angiogenin internalisation by primary astrocytes in culture. Syndecan 4 contribution to both bFGF signalling and endocytosis is well established (Deguchi et al., 2002; Tkachenko and Simons, 2002; Berry et al., 2003; Zhang et al., 2003; Tkachenko et al., 2004; Leadbeater et al., 2006). Initial colocalisation studies showed overlapping angiogenin and syndecan 4 staining in cytoplasmic vesicles, but it turned out to be very difficult to coimmunoprecipitate the two proteins from astrocyte lysates. Optimisation of the protocol by adjusting the salt concentration of the buffers so they resembled Neurobasal medium failed, even though it had been observed that increasing extracellular sodium interfered with angiogenin uptake most likely by preventing syndecan 4 binding on the plasma membrane. Furthermore, syndecan 4 exists in large complexes at the plasma membrane of cells and these might be difficult to catch by precipitation. Another possible explanation could be insufficient specificity of the polyclonal goat anti-angiogenin antibody used or the fact that protein AIG agarose is not ideal for catching goat IgG molecules.

Therefore, the in situ Proximity Ligation Assay was chosen to confirm the interaction of the two proteins within primary astrocytes. This technique does not disrupt cellular integrity as does coimmunoprecipitation, thus it is possible to gain insights into the localisation of interaction sites within cells. Like coimmunoprecipitation, this assay does not exclude the possibility that angiogenin and syndecan 4 may not bind directly but involving other proteins in a complex, but they have to be within 40 nm distance to give a positive signal. Every single spot produced by the assay supposedly corresponds to a single interaction site, as the rolling cycle amplification performed is a very powerful means of signal amplification. Quantification of the spots detected with the aid of the Blob finder software provided by Olink was performed on epifluorescent images using a very high threshold for signal detection, accordingly the true number of interaction sites within a cell is more likely underestimated. Epifluorescent images were chosen for analysis due to rapid image acquisition, however these images displayed blurred areas that could not be quantified. Therefore, it was concluded that angiogenin and syndecan 4 interact specifically within astrocytes possibly as part of a receptor complex, which may include actin as a coreceptor (Hu et al., 1993).

As astrocytes normally express syndecan 4 on their plasma membrane (Hsueh and Sheng, 1999), protein expression was suppressed by shRNA technology. Four commercially available syndecan 4 shRNA constructs coexpressing GFP targeting murine syndecan 4 were purchased from Origene (www.origene.com) in a kit together with the appropriate empty GFP-expressing vector and a scrambled control vector. Transient transfection of primary cortical astrocyte cultures using Lipofectamine 2000 only yielded a transfection efficiency of around 10 %. Additionally, effective gene silencing strongly depended on the expression level of the constructs making it impossible to prove syndecan 4 knock down by Western Blotting. Generally, for efficient gene silencing it was recommended by the manufacturer to collect cell samples for protein quantification after 72 h. Primary astrocyte cultures seeded on poly-L-Ornithine coated coverslips were transfected 24 h after seeding and 72 h later it was observed that many cells had detached. This could be due to the transfection procedure or the actual downregulation of syndecan 4 expression. Syndecans are very important for cell adhesion in general (reviewed by Morgan et al., 2007) and in particular for binding to poly-L-Ornithine or -Lysine in cell culture (Massia and Hubbell, 1992). Therefore, it was impossible to quantify syndecan 4 knock down and angiogenin uptake at any later time points than 48 h after transfection. Nevertheless, reduction of angiogenin internalisation by primary astrocyte cultures transfected with the shRNA constructs and control plasmids was quantified on the basis of randomly taken pseudoconfocal images and tested significant in three experiments. The observation that even the scrambled sequence slightly reduced angiogenin uptake could be explained by changes in cell physiology induced by the transfection procedure or overexpression of foreign protein.To test whether an additional protein was required to induce angiogenin internalisation by cell types other 129 than astrocytes which do not express syndecan 4, NSC34 cells were transfected with tGFP-tagged syndecan 4. These cells with motoneuron-like characteristics do not normally take up angiogenin into vesicles in culture. Syndecan 4 overexpression induced vesicular angiogenin staining, however quantification of the observed induction of internalisation proved to be very difficult, as NSC34 cells do not adhere to poly-L-Ornithine coated coverslips very well. The cells do not spread out on the surface of the coverslip but instead form cell clusters. These cell clusters easily detach and are therefore washed away during cell transfection and immunocytochemical staining. The few cell clusters that remained attached had to be imaged by confocal microscopy as epifluorescent images displayed to much background fluorescence originating from relatively thick samples. Furthermore, angiogenin staining in immunocytochemistry is quite commonly associated with apoptotic cells, causing many bright patches in the angiogenin channel. However, as transfection of murine syndecan 4 was able to induce vesicular uptake of angiogenin in this cell line which otherwise never displayed vesicular angiogenin staining, it was concluded that syndecan 4 expression alone was necessary and sufficient for angiogenin endocytosis.

Taking together the experimental results discussed above it was deduced that angiogenin internalisation by astrocytes in primary cortical cultures is a process requiring for the heparansulfate proteoglycan syndecan 4 as a receptor. Furthermore, proteoglycan-binding of a ligand can support and enhance signalling via a primary receptor or it can lead to endocytosis and degradation of the ligand (reviewed by Belting, 2003). Interestingly, angiogenin has been shown to induce proliferation in endothelial cells (Hu et al., 1997), an effect that was not observed in the experiments performed as part of this study with astrocytes. Possibly, angiogenin endocytosis via its specific receptor leads to completely different cellular responses. To see if any signalling was elicited by angiogenin binding to syndecan 4, activation of the syndecan 4-associated pathways involving PKCa and FAK was examined (Oh et al., 1997b, a; reviewed by Simons and Horowitz, 2001; Wilcox-Adelman et al., 2002a; Keum et al., 2004; Tkachenko et al., 2004). Colocalisation of PKC with angiogenin containing vesicles in astrocytes was observed. The antibody used in this study did not allow for distinction 130 between any particular PKC subtypes, but all published reports showed direct activation of only PKCa by syndecan 4 oligomerisation (Oh et al., 1997b, a; Oh et al., 1998; Keum et al., 2004). It had been described that angiogenin uptake by endothelial cells triggers MAPK signalling (Liu et al., 2001), however involvement of PKC instead of MAPK in astrocytes may also explain the different effects of angiogenin on the two cell types.

lntracellular signalling in response to angiogenin endocytosis by astrocytes and its intracellular activity regarding RNA metabolism remain to be investigated further, however the cell surface receptor for angiogenin uptake by primary astrocytes is the heparansulfate proteoglycan syndecan 4. 4. Chapter ll

Angiogenin Modification of the Astrocyte Secretome

4.1 Chapter IIA

SlLAC Labelling of Primary Astrocytes

4. I.I Introduction

4.1.I Mass Spectrometry

In recent years, evidence has been accumulating underlining non-cell autonomous effects influencing neurodegeneration. As detailed in the introduction (pg. lo), the contribution of cells surrounding motoneurons to disease progression and phenotype in ALS has been the subject of many studies. With the development of modern, very sensitive mass spectrometers (MS), even complex protein mixtures such as the entire set of proteins secreted by cell lines in culture under given conditions can be analysed, which is referred to as the secretome. Several different quantitative proteomics approaches are available to study changes in the pattern of secreted proteins. The following section will explain principles of mass spectrometry and sample preparation.

Mass spectrometers require for the following three major parts: an ion source with optics, the actual mass analyser and a detector (reviewed by Yates et al., 2009).

With the introduction of soft ionisation methods allowing for mass spectrometric analysis of proteins and peptides avoiding degradation (so- called in-source fragmentation), it has become possible to study the protein 132 composition of complex biological samples. One such technique is called matrix-assisted laser desorption ionisation, which generates singly charged ions but results are strongly dependent on sample preparation. Alternatively, electrospray ionisation is used to generate multiply charged ions from solution by applying high voltage between the end of the separation pipeline and the inlet of the mass spectrometer (reviewed by Yates et al., 2009).

The mass analysers can store ions before separating them depending on their mass-to-charge ratios (mlz). Ion separation is achieved by either measuring the ion's time of flight or on the basis of the mlz resonance frequency in ion trap instruments. The latter are best suited for identifying proteins in complex samples as they allow for fast scanning rates and still achieve high sensitivity. The LTQ (Linear trap quadrupole) Orbitrap used in this study consists of a linear ion trap to ensure fast and sensitive sample analysis coupled to an Orbitrap: lons oscillate around a central electrode due to electrostatic fields generated by the instrument (Figure 30), a mass analyser originally developed by Makarov (2000; Hardman and Makarov, 2003). The signal obtained is converted into a mass-to-charge spectrum by a fast Fourier transform algorithm. This type of ion trap is characterised by its high resolution and mass accuracy and can be used to analyse intact proteins ranging from 3 to 150 kDa. (reviewed by Hu et al., 2005; and Perry et al., 2008; and Yates et al., 2009).

Figure 30: View into the Orbitrap mass analyser. lons are injected into the Orbitrap perpen- dicular to the long axis of the Orbitrap (z-axis). A; the ions are not injected at the equator, they start harmonic oscillations around the central electrode in z-direction. The oscillation frequency is independent of injected ion velocity or radius but depends only on the ion's mlz ratio and the potential between the electrodes (Hu et al., 2005). Nevertheless, sample complexity needs to be reduced prior to mass spectrometry when analysing biological protein mixtures as high protein numbers and extreme range of protein concentrations make mass spectra impossible to interpret otherwise. Protein mixtures can be separated by gel electrophoresis using two dimensions if no further separation step is included, however if peptides are later subjected to liquid chromatography, one dimension is sufficient. The intact proteins are then proteolytically digested using enzymes like trypsin generating peptides at known cleavage sites. For this study, the peptides were afterwards separated by reversed phase high-performance liquid chromatography using a capillary column containing C18 for optimal peptide analysis. Peptides were eluted from the column by increasing the acetonitrile concentration in the aqueous buffer and injected straight into the mass spectrometer by electrospray ionisation. The LTQ Orbitrap can be used to acquire MSIMS scans with the Orbitrap performing full MS scans of intact peptides, while the LTQ analyses peptide fragment ions generated by collision-induced dissociation (CID) to determine the amino acid sequences of the most abundant peptides (reviewed by Yates et al., 2009; and Makarov and Scigelova, 2010).

4.1 .I.2 Stable lsotope Labelling by Amino Acids in Cell Culture

Besides obtaining qualitative information about the proteins present in a sample, it is often desirable to achieve absolute or relative quantification, however mass spectrometry is not quantitative in itself. Due to sample variability, it is difficult to obtain sufficient sensitivity when performing relative quantification without using labelled samples (Old et al., 2005). Still, it is possible to compare peak ratios for analogous isotopes. Several labelling strategies have been developed involving either chemical incorporation for example of isotope-coded affinity tags and isobaric tags or biological incorporation using '=N-labelling or by supplementing essential amino acids labelled with stable heavy isotopes of Carbon, Nitrogen or Hydrogen (Deuterium). The latter technique is referred to as Stable lsotope Labelling by Amino acids in Cell culture (SILAC) and was originally introduced using deuterated L-Leucine (Ong et at., 2002; reviewed by Ong et al., 2003). 134 For SlLAC analysis, cell cultures are grown in parallel in normal medium and in medium substituted with heavy isotope-labelled amino acids (Figure 31). Essential amino acids are used as this ensures the amino acids cannot be synthesised from non-labelled compounds present in cell culture. Labelling L- Arginine and L-Lysine, the two amino acids after which trypsin cleaves, ensures that every peptide generated contains at least one labelled amino acid. For cell lines 100 Oh incorporation can be achieved and no adverse effects have been observed in cell cultures. All further processing steps required in preparation for mass spectrometry are then performed on the samples derived from the two cultures mixed straight after harvesting, which greatly improves accuracy of quantification. Furthermore, this technique requires for less labelled protein compared to chemical labelling procedures (reviewed by Ong et al., 2003; and Han et al., 2008).

-Heavy Light - 1. i Figure 31: Schematic representation of SllAC labelling. Cell cultures are grown in / medium substituted with heavy isotope- labelled amino acids or normal medium for several passages. Samples from both cell cultures can be mixed straight after harvesting and subjected to mass spectrometry.

mlz

4.1 .I.3 Analysis of Mass Spectra

The resulting mass spectra generated are very difficult to analyse and require for highly specific software solutions. The mass spectra contain information about peptide mass and intensity (Figure 31) and peptides can be identified by comparing the MS/MS spectra and a sequence database. However, interpretation of mass spectra is complicated as peptides may have different charges or several peptides possibly coelute from the liquid chromatography column. Labelling results in detection of a SlLAC pair or triplet (for double 135 labelling) of peaks with defined difference in mass. The MaxQuant software (Cox and Mann, 2008) used in this study identifies isotope patterns resulting from labelling.

Peptide peaks are then subjected to database search of the fragmentation (MSIMS) spectrum by the Andromeda search engine integrated in the MaxQuant software (Cox et al., 201 I), taking labelling as fixed modification into account. A first search of the fragment spectra on a database containing common contaminants and abundant proteins of the species under investigation is carried out to recalibrate the obtained spectra. The database search is performed using both species-specific, true protein sequences and reversed nonsense sequences. With the MaxQuant software, identified peptides are accepted based on their false discovery rate (FDR), this means if less than I % false hits are retained in the list. The accepted peptides are subsequently assembled into proteins while allowing for an FDR of 1 % at the protein level. A protein is considered successfully identified only if at least two peptides are detected and one of them has to display a unique peptide sequence. Search parameters can be adjusted depending on the exact experimental protocol used, including posttranslational modifications or alternative labelling (Cox and Mann, 2008; Cox et al., 2009).

First of all, efficient incorporation of the labelled amino acids by primary, murine astrocytes was assessed and the treatment protocol optimised to minimise toxic effects on astrocytes.

4.1.2 Results

4.1.2.1 Incorporation of SlLAC Amino Acids

Results obtained using quantitative mass spectrometry are strongly influenced by the labelling technique and depend on label incorporation. Using primary astrocyte cultures, it was therefore necessary to first estimate the incorporation of the labelled amino acids L-Arginine and L-Lysine. Only one study has thus far used SlLAC labelling in murine, primary astrocytes 136 and the authors reported 98 % incorporation after three weeks in labelling medium (Greco et al., 2010). Routine astrocyte preparation had shown that primary astrocytes seeded in T75 flasks derived from 3 cortices reached optimal confluence and viability after two weeks in culture. To be able to include a third treatment condition, the incorporation of both heavy- and medium-labelled L-Arginine and L-Lysine was tested: Heavy L-Lysine contains six I3cand two '=N atoms (Lys8 (L-[I 3C6-I 5N2] Lysine)), while the medium version only contains four deuterium instead of hydrogen (Lys4 (L-[2H4] Lysine)) resulting in a mass difference of four units. Likewise, heavy

L-Arginine contains six I3c and four 15~atoms (ArglO (L-[U-13C6-15N41 Arginine)) and the medium version only six I3c(Arg6 (L-[U-13C61 Arginine)), which also shifts its mass by four units.

The incorporation of labelled amino acids was assessed after one and two weeks in culture on proteins derived from three selected bands: Both a very strong and a faint band were chosen and the three bands picked were distributed across the entire length of the lane. To be able to compare the samples collected after one or two weeks in culture, the ratio of labelled versus unlabelled protein version is depicted on a log2 scale with the values after one and two weeks along the x-axis and y-axis, respectively. This comparison of the ratios of labelled and unlabelled proteins in the samples revealed that there was no difference observable between heavy and medium labelling (Figure 32A and B) as indicated by similar overall protein distribution. There was also little change detectable between labelling for one or two weeks, as proteins identified deviated little from the centre. It was thus chosen to perform treatment after two weeks in culture based on morphology and viability. log2 Fold change (week 1) B

0 I 2 3 4 5 6 7 log2 Fold change (week 1)

Figure 32: Incorporation of labelled amino acids by primary astrocytes after one or two weeks in culture does not change. Primary cortical astrocytes were cultured for one or two weeks in SlLAC DMEM containing heavy (A) or medium (B) labelled L-Arginine and L-Lysine after tissue preparation. Conditioned medium was collected for 24 h, precipitated, proteins electrophoretically separated and trypsin digested prior to mass spectrometric analysis in an LTQ Orbitrap XL. Results are depicted as log2 ratio of labelled versus unlabelled protein versions after one or two weeks (x- and y-axis, respectively). 4.1.2.2 Treatment Protocol Optimisation

The next step in establishing optimal conditions for SILAC labelling was to optimise the exact treatment protocol. As detailed in the materials and methods section (see pg. 38), astrocytes were routinely cultured in DMEM supplemented with 10 % serum, Glutamine and antibiotics. However, it was observed that angiogenin was only endocytosed by astrocytes in Neurobasal medium (see pg. 105). Accordingly, investigating the response of astrocytes to treatment with angiogenin was only possible in Neurobasal medium. This medium however, is not commercially available without the essential amino acids L-Arginine and L-Lysine, it was therefore decided to optimise the treatment paradigm using standard Neurobasal medium. Previous studies establishing angiogeninls neuroprotective properties were performed by treating astrocytes with angiogenin for 6 h, replacing the medium and leaving the cells to condition the medium for another 18 h prior to application to motoneurons.

It was decided to treat astrocytes with angiogenin in Neurobasal and then replace the medium with fresh serum-depleted SILAC DMEM for conditioning. Three treatment protocols were tested and labelling was determined: Cell culture preparation in SILAC DMEM with one medium change at 7 DIV. Treatment with angiogenin on day 14 in culture and conditioning of serum-depleted SILAC DMEM over night. Secondly, an additional medium change was introduced at 13 DIV to prevent the amino acid-starved astrocytes from taking up unlabelled Arginine and Lysine from the Neurobasal after 7 DIV without medium change. In a third condition, it was tested whether supplementing Neurobasal with labelled amino acids on top of the normal amino acids would improve labelling further. Analysing the mass spectrometry data obtained from three bands of the conditioned medium showed that the additional medium change at 13 DIV greatly improved labelling as evidenced by a shift of the light blue spots to higher ratios of labelled versus unlabelled versions (92 % median incorporation versus 95 %; x-axis,. Figure 33). Despite the observation that adding labelled amino acids had no adverse effects on astrocyte viability, little further shift of the red triangles towards higher ratios of labelled proteins was detectable. aA

@ A + Protocol 2 A Protocol 3

#%A* AA4'* - t4 -= ***aA. .& 0' - .rgd

+**AA~$ A r, AA+. * 60

log2 Fold change (Forward ratio HIL)

Treatment Protocol I Protocol 2 A Protocol 3 Medium change at 13 DIV NB6h Labelled amino acids added to NB Median incorporation

Figure 33: Label incorporation is greatly improved by including one additional medium change prior to treatment for medium conditioning. Treatment of primary astrocytes with angiogenin was performed after two weeks in culture in standard, serum-free Neurobasal medium (NB) for 6 h before medium conditioning for 18 h in serum-free SlLAC DMEM (protocol 1). One additional medium change with full SlLAC DMEM was included for protocol 2 and serum-free Neurobasal medium was supplemented with labelled amino acids (protocol 3). Conditioned medium was precipitated, proteins were separated and digested before analysing the samples in an LTQ Orbitrap XL mass spectrometer. Results are depicted as log2 ratio of labelled versus unlabelled protein versions (HIL; x-axis) against the log10 intensity detected by mass spectrometry. Median label incorporation rate is indicated in the bottom line of table. Following the observations detailed above, all treatments to collect astrocyte conditioned medium were therefore performed comparing only two conditions, heavy and medium labelling, as the incorporation efficiency was not sufficient to allow for comparison with the unlabelled, light versions. Too many proteins did not incorporate the label such that their heavyllight ratios were higher than 8 corresponding to an incorporation of 90 % (Figure 33; previous page), which would have been required to include the third treatment condition. Furthermore, in the treatment scheme one additional medium change was inserted at 13 DIV, however it was decided not to supplement Neurobasal medium with labelled amino acids, as the improvement was only minor. Astrocytes were thus washed six times with PBS to remove serum proteins and treated with recombinant human angiogenin (heavy) or its carrier BSA (medium) for 6 h in serum-free Neurobasal medium (Figure 34). After another two washes in PBS, the astrocytes were left to condition corresponding serum-free SlLAC DMEM for 18 h. The medium was collected from both treated flasks, mixed, precipitated and subjected to gel electrophoresis. Gels were stained with colloidal blue staining solution and bands were cut according to the banding pattern indicated in Figure 35. This banding pattern was reproducible across all eight experimental runs, despite cell preparation and medium collection performed at two separate laboratories. Proteins from all twenty bands were subjected to in-gel digestion with trypsin and analysed by mass spectrometry. Heavy Medium 4 Medium change- 7DIV 3 0 0 4 Medium change 13DIV 4 Medium change to Angiogenin 1 Neurobasal for 6 h BSA 6 I -0 L Medium change to 1 --=DMEM (-Ser) for 18 h

Mix conditioned medium from both cell dishes

TCA precipitation of conditioned medium

Gel electrophoresis followed by PAGE Blue staining

Gel bands cut, destained

Proteins digested using trypsin

Peptides injected into LTQ Orbitrap mass spectrometer Figure 34: Optimised treatment scheme of angiogenin stimulation for proteomic analysis of conditioned medium from primary astrocytes. Primary astrocytes were prepared from P2 mouse cortices and placed straight into full SlLAC DMEM for culturing after tissue dissociation. The labelling medium was replaced twice at 7 and 13 DIV before treatment with angiogenin (heavy) or BSA (medium) for 6 h in serum-free Neurobasal. Serum-free (-Ser) SlLAC DMEM was conditioned for 18 h. Conditioned medium was mixed directly after harvesting, cellular debris was removed by centrifugation and the medium precipitated with trichloroacetic acid (TCA). Proteins were separated by gel electrophoresis, stained, cut into bands and proteolytically digested using trypsin. Peptides were extracted from the gel and subjected to mass spectrometric analysis. Labelled and unlabelled peptide versions can be distinguished in the resulting mass spectrum by a characteristic mlz shift and ratios allow for conclusions to be drawn regarding relative protein abundance between treatment conditions (a: double the amount of medium-labelled peptide; b: double the amount of heavy-labelled peptide; c: equal amounts of both labelled peptide versions; d: poor labelling due to large excess of unlabelled, light peptide).

Figure 35: Banding pattern displayed by proteins contained in conditioned medium of primary astrocytes. Gel electrophoretic protein separation of primary astrocyte conditioned medium followed by staining with colloidal blue resulted in the depicted staining pattern (left). The entire lane was cut into 20 bands according to the indicated scheme (centre). Every band was processed for mass spectrometric analysis by trypsin digestion and peptide extraction. Figure shows one representative of eight gels, all of which stained very similarly. 4.1.3 Discussion

Careful optimisation of the experimental protocol with regard to both culture preparation and treatment was necessary, as the study was not performed on immortalised cell lines but primary cell cultures that may be more sensitive to experimental manipulation. Only one study has thus far investigated the astrocyte secretome using SlLAC labelling and the authors reported 98 % of incorporation of the label (Greco et al., 2010). Labelling efficiency strongly depends on protein turnover by the cells, which is majorly influenced by the mitotic index of a cell culture. SlLAC labelling of cell lines is normally quicker, as the immortalised cells usually divide more frequently compared to primary cells. However, primary astrocyte cultures prepared from neonatal mice or rats still retain their ability to proliferate (reviewed by Kimelberg, 1983). The current study was performed on primary astrocyte cultures without further passaging, to avoid unnecessary handling and to reduce the growth of contaminating cells that may proliferate more quickly after passaging. Furthermore, treatment was intended to be performed as early as possible after culture preparation to prevent changes in astrocyte phenotype with culture duration or due to labelling.

Routine morphological observation of the astrocyte cultures did not reveal any changes with labelling, it was thus assumed that labelling did not greatly alter astrocyte physiology. Label incorporation was not sufficient to allow for comparison of three treatment conditions in one mass spectrometry experiment, as some proteins did not display sufficient turnover to ensure optimal label incorporation. There was little change in label incorporation with extending the culture period from one to two weeks, thus it was decided not to attempt to further improve labelling by increasing culture duration. Instead, only two conditions were compared by double labelling with heavy- and medium-labelled amino acids.

The treatment scheme was adjusted to meet two requirements: Ensure good label incorporation for mass spectrometry and to recapitulate previously used treatment schemes for conditioning medium. Due to technical limitations, angiogenin treatment had to be performed in standard Neurobasal containing unlabelled amino acids. However, motoneuron protection assays were performed using astrocyte conditioned medium that was collected after the astrocytes had been treated for 6 h with angiogenin. The effects of this treatment period on label incorporation were difficult to estimate and therefore experimentally tested. After one week in culture without medium change, it was assumed that cells would have depleted the medium of most essential amino acids, thus boosting amino acid supply prior to angiogenin treatment greatly improved labelling. However, additionally supplementing Neurobasal with labelled amino acids did not lead to major improvement of labelling. L-Arginine has been reported to stimulate tumour cell proliferation (reviewed by Szende et al., 2001), however no effects on normal astrocyte morphology or proliferation were observed by routine examination. Furthermore, a problem commonly observed in SlLAC labelling experiments is the conversion of labelled L-Arginine to L-Proline, which influences mass spectrometry results (reviewed by Marcilla et al., 201 1 ). As the improvement of label incorporation was so little, it was considered not to be necessary to supplement Neurobasal with labelled amino acids to avoid changing the proliferative state of the cell cultures or to induce L-Arginine conversion by supplying it in excess. Consequently, it was decided to perform treatment in standard serum-free Neurobasal.

During experimental preparation of conditioned medium, the first read-out of sample quality is the banding pattern of the gel after gel electrophoresis and staining. Major contamination with BSA would be easily detectable as a prominent band at 66 kDa, however it was never observed in this study as sample preparation included eight washing steps in PBS together with the treatment in serum-free Neurobasal prior to medium collection. The banding pattern observed for all of the full secretome screens was very similar. Sample preparation for the first secretome screen was carried out at the lab in Dublin, while comparative analysis between angiogenin and BSA treatment was performed in Montpellier. Accordingly, cell cultures were prepared from different animals and grown in different environment. The observation that the banding pattern was so reproducible indicated robustness of the experimental protocol used including tissue preparation and medium collection for mass spectrometry.

The next step was to functionally validate the model used by thoroughly characterising the proteins secreted by untreated astrocytes and comparing it to existing data published on the secretome of primary astrocytes in culture. Chapter IIB

The Astrocyte Secretome

4.2.1 Introduction

4.2.1 .I Secretory Routes

Mammalian cells are able to secrete proteins by various mechanisms depending on the fate and purpose of the protein to be secreted. Conventional protein secretion requires for the protein to contain an amino terminal signal sequence usually consisting of positively charged amino acids followed by eight or more nonpolar amino acids. This sequence targets the protein for cotranslational import into the ER. The protein is then routed from the ER to the Golgi apparatus where it is targeted for secretion unless it contains a retention signal at its carboxy terminus. During sorting, the signal sequence is removed and proteins may be subjected to postranslational modification such as proteolytic processing and glycosylation. Classically, three types of secretory pathways are distinguished (Figure 36): Constitutive secretion is considered to be the default pathway as it does not require any additional signal. Alternatively, proteins can be targeted from the Golgi apparatus to lysosomes or destined for regulated secretion (summarised by Lodish, 2000; and Alberts, 2002). Figure 36: Protein sorting for conventional secretion. By default, proteins are constitutively secreted unless they contain a signal like mannose 6-phosphate targeting to the lysosome or other signals for specific secretory vesicles. The latter are only released from the cell in response to stimulation (Alberts, 2002).

However, it has been observed that several proteins are secreted following non-classical routes, among which four principal mechanisms are discussed (Figure 37): Proteins can be directly translocated from the cytoplasm across the plasma membrane via transporter proteins. Secretory lysosomes are loaded with cargo proteins and release these by fusing with the plasma membrane. Extracellular vesicles are generated by the fusion of multivesicular bodies with the plasma membrane generating exosomes. Alternatively, exovesicles can directly be formed by membrane blebbing from the plasma membrane. Several different types of proteins that are normally found extracellularly have been shown to be secreted by unconventional routes including bFGF, aFGF, lectins, interleukin-1P and macrophage migration inhibitory factor. Interestingly, proteins with well-established intracellular function can be released upon stimulation such as thioredoxin, which is required to maintain redox balance, or the nuclear protein high- mobility group box 1 protein. Additionally, several proteins containing signal sequences are secreted without requiring sorting through the Golgi apparatus like the cystic fibrosis transmembrane conductance regulator (reviewed by Nickel, 2005; and Nickel and Seedorf, 2008; and Nickel and Rabouille, 2009). Plasma membrane-

Unconventlonal secretory cargo molecule Cytoplasm

Figure 37: Unconventional protein secretion. Proteins can be secreted from secretory lysosomes (I),directly via transporters across the plasma membrane (2), by fusion of multivesicular bodies with the plasma membrane releasing exosomes (3),or by membrane blebbing generating exovesicles (4) (Nickel, 2005).

Prediction tools are available to identify both classically and unconventionally secreted proteins based on sequence similarities. These are commonly used to validate mass spectrometry results from secretome screens, as these often contain proteins not normally known to exhibit extracellular functions (see below).

4.2.1.2 Astrocyte-Secreted Proteins

Proteomics approaches have lately been used to identify biomarkers for several different diseases, but due to technical limitations caused by abundant serum proteins for example, this has proven to be very difficult. To avoid the problems arising from the use of clinical samples, many studies have been performed using cell lines, primary cells or stem cells to collect conditioned medium for analysis by mass spectrometry (reviewed by Dowling and Clynes, 201 1; and Skalnikova et al., 201 1).

Recently, in an attempt to characterise the proteins secreted by primary astrocytes to gain further insights into how these cells regulate homeostasis 149 in the central nervous system and support surrounding neurons, several studies using mass spectrometry have been performed. The first study identified over thirty proteins in the supernatant of primary mouse astrocytes derived from four different brain regions, many of which display antioxidant or proteolytic properties (Lafon-Cazal et al., 2003). This protein list was extended to forty proteins by the same group using a labelling approach (Delcourt et al., 2005). In 2009 and 2010, four further studies were published characterising the secretome of primary astrocytes: 290 proteins were identified in the supernatant of murine, primary cortical astrocytes after seven days of conditioning of the medium (Keene et al., 2009). To ascertain extracellular protein localisation, gene ontology (GO) analysis was performed and the two prediction tools Protein Prowler and SecretomeP were used to identify classically and unconventionally secreted proteins, repectively. A similar study performed on primary rat astrocytes identified 302 proteins in the astrocyte supernatant collected for 24 h, of which 133 display confirmed extracellular localisation by GO anlaysis (Moore et al., 2009). An optimised protocol for collecting conditioned medium from primary, murine astrocytes for 24 h was used by Dowell (2009), who identified 187 extracellular proteins by GO analysis, SignalP and SecretomeP prediction from a list of 423 total proteins. The same lab used a different approach by determining the fold enrichment of proteins in the supernatant after 7 d compared to the whole cell lysate to more specifically identify 92 secreted proteins (Greco et al., 201 0).

The studies summarised above all identified overlapping sets of proteins and many used computational tools to confirm results. In conclusion, they all support the initial observation that astrocytes majorly contribute to ECM formation and modification by proteases and highlight their role in growth and development, immune responses and oxidative stress in the brain. To evaluate the cell culture model for the current study and to rule out possible effects of SlLAC labelling on primary astrocytes, the complete astrocyte secretome was first analysed and compared to the results obtained in the studies mentioned above. 4.2.2 Results

4.2.2.1 Comparison with Published Data

In an initial experiment, the treatment protocol used was validated and compared to previous studies. Four samples of conditioned medium from the astrocyte cultures treated in the same manner as for angiogenin stimulation were analysed by mass spectrometry using an LTQ Orbitrap Velos and secreted proteins were determined. In total, 1431 proteins were identified and these results were compared to the published protein lists. Of the secreted proteins detected by Keene (2009), 81 % were also found in the conditioned medium of astrocytes under the treatment protocol used for this study (Figure 38A). Comparing the identified proteins with Dowell's results (2009), 68 % of the reported proteins secreted by astrocytes were also found in the present study (Figure 38B). The last study by Greco (2010) identified 92 astrocyte- secreted proteins, of which 89 % were also detected in the current mass spectrometry study (Figure 38D). These comparisons were performed without clearing the complete list of detected proteins of intracellular proteins, which is one reason for the large excess of proteins currently identified. Of the complete lists of proteins detected by Dowell and Greco, 61 % and 81 % were also identified is this study, respectively (Figure 38C and E). As the majority of previously reported astrocyte-secreted proteins from both the cleared and uncleared protein lists were also present on the current list, it was concluded that the treatment paradigm was valid to study the astrocyte secretome, despite the fact that none of the previous studies used the same culture preparation technique or treatment conditions. Figure 38: The majority astrocyte-secreted proteins described in previous publications are detected in the present screen. Venn-diagrams illustrating the overlap ( ) between astrocyte-secreted proteins detected in this screen (blue) compared to previous publications (red) by Keene (2009), Dowell (2009) and Greco (2010). Comparison with extracellular proteins identified by Keene (A), Dowell (B) and Greco (D) or the complete lists published by Dowell (C)and Greco (E). 4.2.2.2 Gene Ontology Analysis

The studies previously performed on the astrocyte secretome (see pg. 149) all reported many intracellular proteins found in the astrocyte supernatant despite filtering or centrifuging the conditioned medium after harvesting and reporting little cell death in the cultures used. It was therefore decided to perform gene ontology analysis (see pg. 71) on the proteins detected in the supernatant to get a better overview of how the identified proteins fit in the three gene ontology groups commonly distinguished: Biological Process, Cellular Component and Molecular Function (Figure 39; A, B, C, respectively). These annotations are based on databases generated by an international bioinformatics initiative aiming at standardising the representation of genes and their products (Ashburner et al., 2000). Gene ontology analysis groups the proteins of a list depending on their characteristics, thus proteins may be filed belonging to several annotations. The resulting annotations allow for conclusions to be drawn regarding the overrepresented functions or processes in the list of proteins of interest.

Within the Biological Process grouping (Figure 39A), most of the detected proteins are generally involved in cellular or metabolic processes, however many also have roles in biological regulation, development, localisation and responses to stimuli. This highlights the importance of astrocytes in brain homeostasis. Looking at the Cellular Components that the identified proteins belong to (Figure 39B), the third largest group are classically extracellular proteins (Extracellular region part, Extracellular region, Extracellular matrix). However, the majority of proteins are not surprisingly part of the cell (Cell part) and mostly associated with organelles (Organelle, Organelle part). When taking Molecular Function into account (Figure 39C), most proteins bind other components (Binding) and display catalytic activity. Still, astrocytes also secrete proteins that regulate enzymes, transduce signals or are structural components (Enzyme regulator activity, Signal transducer activity, Structural molecule activity). This analysis was also performed to better understand the importance of the changes in secretion induced by angiogenin. ICellular process Metabolic process

I Biological regulation

I Developmental process Multicellular organismal process Localisation

tu Response to stimulus Other

EI Not found

Cell part Organelle Organelle part Protein complex Extracellular region part Extracellular region a Extracellular matrix r Other r Not found

IBinding

ICatalytic activity Molecular function Enzyme regulator activity w Signal transducer activity

IStructural molecule activity

I Other Not found

Figure 39: Gene ontology annotation of identified astrocyte-secreted proteins. The complete list of proteins identified in the supernatant of primary astrocytes after 18 h was submitted for gene ontology annotation into the three domains Biological Process (A), Cellular Component (B) and Molecular Function (C) with the aid of a GO tools package in R (Paquet and Yang, 2007). Data are represented as percent of total positive annotations found within the analysed list of lead proteins identified from the match group list generated by MaxQuant. 4.2.2.3 KEGG Analysis of Enriched Pathways

Another way to interpret large data sets obtained in mass spectrometry screens is to subject the data to functional annotation in a pathway-sorted manner. One modelling and simulation tool commonly used is the KEGG (Kyoto Encyclopedia of Genes and Genomes) collection of originally three main online databases, which are Pathway, Genes and Ligand (Kanehisa and Goto, 2000). The databases available are continuously expanded and new databases have been added. With the aid of the DAVID (Database for Annotation, Visualisation and Integrated Discovery) bioinformatics resources website (Huang da et al., 2009b, a), the list of identified proteins was subjected to functional annotation and 48 Oh of the proteins were contained in the KEGG pathway database. Data are presented as the percentage of identified proteins assigned to the top ten scoring pathways, as determined by a modified Fisher's exact test, and the FDR is indicated at each bar. The three most significantly enriched pathways derived from astrocyte-secreted proteins are the proteasome, the lysosome and the ribosome (Figure 40). Interestingly, the astrocyte secretome also contains many proteins involved in focal adhesions and interactions between the ECM and receptors (ECM- Receptor interaction), which further validates the model used and shows that the samples were of appropriate quality to analyse secreted proteins relevant to astrocyte function. In accordance with previous reports which detected antioxidant proteins secreted by astrocytes (Lafon-Cazal et al., 2003), they also secrete proteins involved in glutathione metabolism, underlining their role in oxidative homeostasis.

KEGG pathway data are normally represented in pathway maps, which highlight all the proteins that were present in any list submitted for analysis. The ECM-receptor interaction (Figure 41; pg. 156) map is depicted, as it shows that in fact most of the extracellular interaction partners of integrins, important mediators of cell adhesion, were found to be secreted by astrocytes. This scheme also illustrates several of the proteins detected that are involved in focal adhesion formation, including the previously described syndecan 4. These molecules contribute majorly to formation of the ECM and are thus important for functional brain architecture. Proteasome 1- 1.21E-19 Lysosome J 7.16~-20 Ribosome 6.44~-I5 Pentose phosphate pathway Focal adhesion 7.47~-05 ECM-Receptor interaction 6.27E-05 Other glycan degradation 5.92E-05 Glutathione metabolism 8.89E-05 Spliceosome - 3.00~-04 Prion diseases 4.07~-04 w 1 I 4 2 3 % Identified proteins

Figure 40: KEGG pathway analysis of identified astrocyte proteins. The complete list of proteins identified in the supernatant of primary astrocytes after 18 h was submitted for functional annotation with the aid of the DAVID bioinformatics website (Huang da et al., 2009b, a). The ten most significantly enriched KEGG (Kanehisa and Goto, 2000) pathways determined by a modified Fisher's exact test are depicted with the percent of proteins assigned to the corresponding pathway within the 690 proteins included in the KEGG pathway database. The numbers given next to the bars are the Benjamini-Hochberg FDR values (1995).

Figure 41: KEGG pathway representation of proteins involved in ECM- receptor interactions detected in the current screen. The scheme shows all proteins involved in ECM-receptor interactions identified from the submitted list of proteins highlighted (red) that are also found in this KEGG (Kanehisa and Goto, 2000) pathway database. Non-highlighted proteins are non-identified interaction partners (black). Abbreviations: BSP: lntegrin Binding Sialoprotein; Chad: Chondroadherin; DG: Dystroglycan; HA: Hyaluronic Acid; Ig-SF: immunoglobulin Superfamily; OPN: Secreted Phosphoprotein 1; RHAMM: Hyaluronan-mediated Motility Receptor; SV2: Synaptic Vesicle Glycoprotein 2c; THBS: Thrombospondin; VLA: Very Large Antigen; VWF: Von Willebrand Factor Homolog See following page. ECM lntegrin ECM lntegrin Leukocyte pratieins ECM Proteo- glycan 4.2.3 Discussion

Prior to this study, six studies have attempted to globally characterise the primary astrocyte secretome using mass spectrometry. Despite the fact that none of the published studies was sensitive enough to capture growth factors secreted by astrocytes, they displayed overlapping results with regard to the extracellular proteins identified (Dowell et al., 2009). Comparison between the publications is hampered by the use of different protein identifiers (such as UniProt accession numbers or Entrez genes) and changing identifiers even when the same source database is used. The UniProt (Universal protein resource) database employed for this study is amended on a regular basis as knowledge increases in a collaboration between three institutes (UniProt Consortium, 201 1b).

For this preliminary screen of astrocyte-secreted proteins, it was decided to dispense with filtering the complete list of over 1400 proteins for exclusively extracellular proteins. With the confirmation that the majority of proteins previously detected in astrocyte supernatant were also identified in the current screen, it was concluded that the experimental procedure was appropriate to study the astrocyte secretome. When comparing the overlap of the lists containing confirmed extracellular proteins and the proteins detected in total in the two most recent studies on the astrocyte secretome (Dowell et al., 2009; Greco et al., 2010), little difference in the percentage of proteins contained in the current study was calculated for either publication. However, only three times less proteins were identified in total, which makes the current list the most comprehensive to date and underlines the sensitivity of the technique used.

In recent years several studies have been performed on unconventional protein release, especially via exosomes, a mechanism allowing for the release of classically intracellular proteins into the extracellular space (reviewed by Keller et al., 2006). Exosomes have also been observed to be released by astrocytes and neurons in culture (Faure et al., 2006), thus the presence of intracellular proteins in the supernatant of primary astrocyte cultures may still reflect true secretion, not just release from dying cells. Staining primary astrocyte cultures with trypan blue in the tissue culture flasks used for medium collection indicated very little naturally occurring cell death and no gross changes in astrocyte morphology were observed by routine examination despite the washing procedure with PBS. Washing astrocytes with PBS has previously been reported to lead to astrogliosis (Dowell et al., 2009), however the protocols used for preparing and maintaining astrocytes differ, thus distinct astrocyte behaviour could be attributed to subtle variation in overall astrocyte physiology.

Gene ontology analysis is commonly performed to extract information from large data sets that are difficult to analyse without the aid of such bioinformatics tools. Analysis performed on the complete set of proteins detected in the supernatant of primary astrocytes was aimed at characterising basal protein secretion in order to be able to more easily detect and interpret treatment-induced changes. With regard to the Biological Processes astrocyte-secreted proteins are involved in, the central role of astrocytes in maintaining homeostasis in the CNS becomes clear, as the major groups are the general annotations of Cellular and Metabolic processes as well as Biological regulation. However, the flexibility and integrative role of astrocytes is also evident as the secreted proteins also contribute to Developmental processes, Localisation and Responses to stimuli. Looking at the subcellular distribution of proteins identified in the astrocyte secretome, a large number are associated with extracellular space, but as indicated above, it is very common to find intracellular proteins in cell culture supernatants and is not necessarily attributable to reduced cell viability in culture. Regarding Molecular Function, the two biggest groups are associated with Binding and Catalytic activity, very general attributes of proteins. However, many proteins are also involved in enzyme regulation and structure formation, which points towards the role of astrocytes in shaping the ECM by secreting metalloproteinases and their inhibitors as well as ECM components. Taken together, gene ontology analysis confirmed the established roles of astrocytes in CNS function and architecture, which are still mimicked in cell culture. Pathway analysis using KEGG showed significant secretion of components involved in protein degradation (proteasome, lysosome), however the identification of lysosomal proteins secreted by cells is not surprising bearing in mind the process of lysosomal secretion detailed above (see pg. 147). Secretion of ribosomal or spliceosomal components could be explained by exosome secretion, as previous proteomics studies on exosomes derived from different cells in culture have reported the presence of splicing factors (Lee et al., 2009) and ribosomal components (Choi et at., 2007; Chavez- Munoz et al., 2009; Mathivanan et al., 2010; Welton et al., 2010). Detection of ECM and focal adhesion proteins by KEGG proves validity of results obtained by the experimental protocol used and further highlights the role of astrocytes in shaping the ECM.

In conclusion, the experimental paradigm used in this study is in good agreement with published data about secreted proteins by various mechanisms and was considered appropriate to study changes in the astrocyte secretome induced by angiogenin stimulation. 4.3 Chapter IIC

Comparative Astrocyte Secretome Analysis

4.3.1 Introduction

4.3.1.1 Studying Astrocyte Secretion under Inflammatory Stimulation

Neurodegenerative diseases like ALS are often associated with inflammatory changes in the CNS and astrocytes may be activated causing reactive gliosis. In addition to treating primary astrocyte cultures with the commonly used drug Brefeldin A, an inhibitor of Golgi-dependent secretion, to confirm extracellular protein localisation (Lafon-Cazal et al., 2003; Dowell et al., 2009; Greco et al., 2010), several of the studies detailed previously (see pg. 149) performed on the astrocyte secretome also attempted to identify changes in the pattern of secreted proteins by astrocytes in response to inflammatory stimuli.

Initially, cultured astrocytes were subjected to LPS, interleukin-I p and TNFa and only select changes in protein secretion were detected based on 2D gel analysis (Lafon-Cazal et al., 2003). The same group performed chemical Lysine labelling to quantify changes in protein secretion in response to LPS stimulation and analysed differential expression of 16 secreted proteins: Half of the proteins analysed were secreted to the same extent as without treatment, while secretion of the second half of proteins was increased by LPS stimulation (Delcourt et al., 2005). The third study on the effects of inflammatory cytokines investigated changes in the astrocyte secretome following treatment with interleukin-lg, interferon-y or TNFa for 24 h and 7 d (Keene et al., 2009). Cell viability remained unchanged, however the astrocytes expectedly displayed morphological changes such as enhanced process formation and secreted inflammatory markers like interleukin-6, NO and prostaglandin ES. More proteins were identified when conditioning for 7d, probably due to basal protein secretion resulting in sufficient 161 accumulation only after prolonged collection periods. This study identified 15 proteins to be present only in control supernatant and twelve proteins that only appeared after cytokine treatment, the most important of which are matrix metalloproteinase-3 (MMP3) and several chemokine ligands.

Taken together, these studies further underline the important role of astrocytes in shaping the environment in the CNS: Several of the proteins regulated in response to inflammatory stimulation are expectedly involved in immune responses, which emphasises the contribution of astrocytes to immune function in the CNS (reviewed by Dong and Benveniste, 2001). Additionally, astrocytes under inflammatory stimulation strongly affect the ECM, which could have severe consequences for neighbouring neurons with regard to scar formation and regeneration.

4.3.1.2 Studying Astrocyte Secretion under Neuronal Stimulation

It has long been established that astrocytes express receptors for neurotransmitters and also respond to their release by neurons (reviewed by Araque et al., 1999; and Perea et al., 2009; and Giaume et al., 201 0). They not only contribute to neurotransmitter clearance (as indicated for the glutamate transporter EAAT2; see pg. 13), but also regulate cerebral blood flow in response to neuronal activity (reviewed by Gordon et al., 2007; and ladecola and Nedergaard, 2007; and Attwell et al., 201 0).

The only proteomic study performed on the secretome of primary rat astrocytes in response to neurotransmitter stimulation was initiated following the observation that astrocytes not only respond to cholinergic stimulation (Araque et al., 2002; Perea and Araque, 2005), but stimulated astrocytes secreted factors that influence neuritogenesis and differentiation of hippocampal neurons (Guizzetti et al., 2008). Astrocyte stimulation was performed for 24 h using the cholinergic agonist carbachol (Moore et al., 2009). The authors identified 32 proteins that were significantly regulated in response to stimulation, 15 of which were upregulated. The list of regulated proteins contains several factors involved in neuritogenesis and again molecules involved in immune responses and modification of the ECM. 162 It is intriguing to observe that astrocytes obviously respond to different types of stimuli by modulating both immune responses and shaping the ECM, which highlights their role in brain homeostasis and architecture. Additionally, the importance of fine tuning the composition of the extracellular environment for normal CNS physiology with regard to signalling and structural molecules becomes clear. A large scale proteomic screen using quantitative mass spectrometry was therefore performed to observe changes in astrocyte secretion in response to angiogenin stimulation to gain further insights into its neuroprotective activity.

4.3.2 Results

4.3.2.1 Secreted Proteins Regulated by Angiogenin

Finally, changes in astrocyte-secreted proteins with angiogenin stimulation in comparison to BSA treatment were analysed. Addition of BSA in control condition was necessary, as recombinant human angiogenin protein was solubilised in presence of 50-fold excess of BSA to ensure proper protein folding in solution and to stabilise the protein. To avoid contamination of mass spectrometry results with common serum proteins, as these could mask any less abundant secreted proteins, cell treatment had to be performed without serum in the medium. Accordingly, BSA was used as a control to distinguish angiogenin-mediated effects from BSA effects, as astrocytes express receptors for albumin (Juurlink and Devon, 1990).

The results from the mass spectrometry screen are shown as log2 ratio of the heavy versus medium protein versions against log10 intensity detected by the mass spectrometer (Figure 42). Accordingly, proteins that are unchanged between the two treatment conditions are clustered at the centre of the graph with log2 ratios close to zero. It was concluded that angiogenin treatment did not cause any changes in the overall secretion profile, as indicated by the fact that most detected proteins clustered along the midline. However, when comparing to BSA treatment, angiogenin stimulated

163 downregulation of secretion of more proteins than upregulation, as there were more proteins with negative log2 ratios. Some of the most significantly regulated proteins are indicated on the graph (the ones chosen for validation are highlighted red), the complete lists of up- and downregulated proteins as tested by significance B testing (Table 13 and Table 14, respectively) confirmed that more proteins were downregulated by angiogenin treatment compared to BSA. Significance B testing was used, as the total list of proteins contained more than 2,000 entries, thus making the introduction of intensity-dependent subgroups for statistical testing feasible. The graph illustrates how the ratios spread less from zero with higher signal intensities, therefore using significance B testing fits better to the overall results obtained (Figure 42). t

.-0 V) C 22 K co-

Plau

6 log2 Ratio HIM

Figure 42: Astrocyte-secreted proteins regulated by angiogenin stimulation. Detected proteins are represented according to their log2 normalised ratio of heavy versus medium protein (Ratio HIM) against the log10 intensity of the mass spectrometric signal. Results of significance B testing are indicated with the aid of a colour code (top right corner): Significantly regulated proteins are represented by red or yellow points. Blue points are proteins not significantly regulated. Selected regulated proteins are assigned with their gene names, names highlighted red are the proteins selected for further validation. Table 13: Significantly upregulated proteins: Proteins are given with the UniProt accession, gene name and full protein name. MaxQuant results obtained for the median normalised heavy versus medium (HIM) protein ratios across all four experimental repeats are given in log2 along with log10 values of median signal intensities. The last column contains the p-values calculated by significance B testing. Table 14: Significantly downregulated proteins: Proteins are given with the UniProt accession, gene name and full protein name. MaxQuant results obtained for the median normalised heavy versus medium (HIM) protein ratios across all four experimental repeats are given in log2 along with log10 values of median signal intensities. The last column contains the p-values calculated by significance B testing.

-- chain -- PO1901 H2-K1 H-2 class I histocompatibility -1.23 7.66 1.67E-06 antigen, K-B alpha chain pp QOVGU4 Vgf MCG18019 -1.20 7.31 3.07E-06

In addition, results from individual significance B testing for regulated proteins following angiogenin treatment between the four experimental repeats was routinely performed to confirm reproducibility between experiments. Graphs from individual experiments are again shown as log2 ratio of heavy versus medium protein versions against log10 signal intensity measured (Figure 43). The proteins selected for further validation are indicated in red to underline overall good reproducibility of experimental results between repeat experiments.

The complete list of regulated proteins was subsequently subjected to more detailed analysis. Initially, extracellular localisation of the regulated proteins was confirmed using the prediction tools SignalP (Bendtsen et al., 2004a) and SecretomeP (Bendtsen et al., 2004b). SignalP predicts the existence of a signal peptide for protein sorting into the ER on the basis of the amino acid sequence. SecretomeP detects pathway-independent features that secreted proteins have in common to predict whether a protein could potentially be secreted even without displaying a classical signal peptide. This analysis confirmed that of the I09 proteins found to be regulated, 88 % are secreted proteins. *Plau .,. .

I, t -4 -2 0 2 4 -4 -2 0 2 4 log2 Ratio HIM log2 Ratio WM

I, I 4 -2 0 2 4 r, -2 o 2 i log2 Ratio WM log2 Ratio HIM

Figure 43: Good reproducibility of detected astrocyte-secreted proteins regulated by angiogenin stimulation between four experimental repeats. Detected proteins are represented according to their log2 normalised ratio of heavy versus medium protein (Ratio HIM) against the log10 intensity of the mass spectrometric signal for all four individual experimental repeats (A to D). Results of significance B testing are indicated with the aid of a colour code (top right corner in graph A): Significantly regulated proteins are represented by red or yellow points. Blue points are proteins not significantly regulated. Proteins selected for validation are highlighted with their gene names. 4.3.2.2 Changes in Functional Annotation with Angiogenin Treatment

As detailed in the previous section (see pg. 153), large lists of proteins can be analysed with the aid of gene ontology annotations, which allow for visualisation of the data and identification of functional groups that are over- or underrepresented. To gain further insights into the impact of angiogenin treatment on these functional categories, the list of secreted proteins was subjected to gene ontology analysis using the BiNGO plugin (Maere et al., 2005) available for the open source software Cytoscape (Shannon et al., 2003). Cytoscape can be used for integrating biomolecular interaction networks from large databases and the BiNGO plugin allows for determination of the significantly overrepresented gene ontology terms in a list of genes. The software generates networks in which the overrepresented terms are displayed in a colour-coded fashion depending on their significance testing according to Benjamini and Hochberg (1995).

Three lists of genes were analysed using BiNGO for all three gene ontology groups: The complete list of regulated genes, the downregulated and the upregulated genes separately. The resulting networks visualising overrepresented Biological Processes (Figure 44A) nicely illustrate how angiogenin globally changes the secretion of proteins involved in responses to cell stimulation, more specifically to stress and biotic stimulation (Response to stress, Response to biotic stimulus). Additionally, many proteins with roles in Cell communication and Cellular component organisation are regulated. Looking at upregulated processes, proteins involved in Multicellular organismal development and Cell proliferation are overrepresented, while the proteins in external stimulation, stress or biotic stimulation are all downregulated (Response to external stimulus, Response to stress, Response to biotic stimulus). When looking at the Cellular Components (Figure 44B) angiogenin-regulated proteins belong to, the most significantly overrepresented are extracellular components, which also appear to be the most upregulated protein annotations (Extracellular region: Extracellular space). Conversely, of the downregulated proteins, some are still associated with mainly intracellular function, despite testing for unconventional secretory routes (see pg. 163). With respect to Molecular Function (Figure 44C), the regulated proteins displaying Binding activities are overrepresented, mainly Receptor binding, but also some Carbohydrate binding. Protein binding is also the most overrepresented function of both, the upregulated and downregulated proteins, however several of the downregulated proteins are also Nucleic acid binding proteins.

Taken together, this analysis confirmed the regulation of extracellular proteins involved in external and stress responses by angiogenin treatment of astrocytes. The regulated proteins in their turn interact with appropriate extracellular binding partners, which may be proteins, carbohydrates or RNA to modify the extracellular environment thereby influencing motoneuron survival. Downregulated Regulated Upregulated

Cellulav Component Cellul ' Tonent T

t Molecular Function ~olecularFunction Moleculpr tunction - -

Figure 44: Functional gene ontology analysis of proteins with significantly regulated secretion by angiogenin treatment of astrocytes. Overrepresented GO terms in the groups Biological Process (A), Cellular Component (B) and Molecular Function (C) of regulated astrocyte- secreted proteins statistically analysed and visualised with the aid of the BiNGO 2.44 plugin (Maere et al., 2005) for the Cytoscape network modelling software (Shannon et al., 2003). Overrepresentation was determined in comparison to the complete mouse genome and statistical testing performed with the aid of the Benjamini & Hochberg FDR correction (1995). Colour key represents range of p-values for significant GO terms. Parental GO terms not testing significant are represented as white circles. Size of circles corresponds to number of members within that term. 4.3.2.3 Validation of Selected Regulated Proteins

To further confirm the validity of the results obtained by mass spectrometry, several proteins were selected for testing by Western blotting (gene names highlighted in the text): Five of the downregulated, namely Histone H4 (H4), interleukin-I receptor antagonist (ILlra), MMP3, collagen lV (Col IV) and SerineIArginine-rich splicing factor I (Srsfl) as well as two upregulated proteins were analysed, urokinase-type plasminogen activator (Plau) and transforming growth factor-P-induced protein ig-h3 (Tgfbi). Additionally, an actin control was included, because the protein was detected in the astrocyte supernatant, but was not regulated with angiogenin stimulation. H4 was picked (log2 ratio HIM -1.09; just not significant), as it has previously been detected in the supernatant of astrocytes, even though it is not predicted to be secreted (Greco et al., 2010). The protein lLlra inhibits interleukin-I binding to the cell-surface receptor (reviewed by Arend and Guthridge, 2000) and interleukin-I has been suggested as a potential therapeutic target for ALS (reviewed by van der Meer and Simon, 2010). MMP3 has previously been observed in the astrocyte secretome in response to cytokine stimulation (Keene et al., 2009), however the role of MMPs in CNS pathology is very complex (reviewed by Rivera et al., 2010; and Kim and Hwang, 201 1). As a representative of the ECM molecules secreted by astrocytes Col lV was chosen, the isoforms of which are expressed in the nervous system in distinct locations and contribute to targeting of developing axons (reviewed by Hubert et al., 2009). The splicing factor Srsfl was investigated, as angiogenin is involved in RNA metabolism and splicing factors have been observed to be secreted in vesicles (Lee et al., 2009). Transfer of RNAs and proteins via exosomes has also been suggested to take place at synapses in the CNS (reviewed by Smalheiser, 2007). The upregulated protein Plau was further investigated, due to angiogenin's role in regulating angiogenesis, which involves plasmin activation (see pg. 25). Tgfbi has been discovered as a protein induced by TGF-P (transforming growth factor+) inhibiting cell attachment (Skonier et al., 1994) and TGF-P signalling is a potential target for ALS therapy (reviewed by Katsuno et al., 201 1 ). The samples for Western blotting were prepared using exactly the same protocol as for mass spectrometry, however the supernatants from angiogenin- and BSA-treated astrocytes could not be mixed, but had to be precipitated and subjected to gel electrophoresis separately. One additional condition was included for Western blotting analysis to be able to estimate the effects of BSA treatment on astrocytes under serum-withdrawal. Proteins were separated on precast gradient gels to visualise all proteins of different molecular weight on the same gel type and to reduce variability. Regulation of secretion by angiogenin was confirmed by Western blotting in three independent experiments for all of the proteins tested except one and representative blots are shown (Figure 45). Col lV was difficult to detect under the conditions used, probably due to denaturation of the epitope recognised by the antibody. Srsfl was the only protein, which displayed too much inter-sample variability to draw any conclusions about its regulation by angiogenin. Nevertheless, angiogenin lead to decreased secretion of H4, ILlra, MMP3 and Col IV compared to BSA, which by itself increased the extracellular amount of these proteins in comparison with untreated cells. Both Plau and Tgfbi were detected also in the untreated control and their secretion was reduced by BSA compared to angiogenin as expected from the SILAC ratios. In conclusion, Western blotting confirmed the mass spectrometry results obtained with regard to the effect of angiogenin on the astrocyte secretome. CM Lysate CM Lysate BSA cc ,n Ang kDa Ang con BSA BSA con Ang kDa Ang con BSA

Figure 45: Western blotting of selected, regulated proteins mostly confirms proteomic SlLAC results. Representative Western blots performed on primary astrocyte conditioned medium (CM; 1'' and 3rd panel) and whole cell lysates (2ndand 4'h panel) probing for the proteins histone H4 (H4), interleukin-I receptor antagonist (ILlra), matrix metalloproteinase-3 (MMP3), collagen IV (Col IV), SerinelArginine-rich splicing factor I (Srsfl), Urokinase-type plasminogen activator (Plau), Transforming growth factor-p- induced protein ig-h3 (Tgfbi) and actin. Astrocyte stimulation for Western blotting included BSA- and angiogenin-treated (Ang) astrocytes as well as untreated control astrocytes (con). Proteins found to be downregulated by angiogenin treatment compared to BSA by SlLAC are depicted in the five top rows, upregulated proteins in the 6thand 7ih row. Actin was included as control protein not regulated by astrocyte treatment with angiogenin. Western blots for all eight proteins were repeated at least three times. 4.3.3 Discussion

Analysing changes in protein secretion by mass spectrometry can be used as a simplified model for biomarker discovery (reviewed by Dowling and Clynes, 201 I),however it is very important to interpret data very carefully despite thorough optimisation of treatment conditions. The observation that angiogenin treatment induced only subtle changes in astrocyte protein secretion, highlighted the specificity of the proteins that were identified to be regulated. Most of the proteins detected by mass spectrometry clustered at log2 ratios close to zero of heavy versus medium protein versions. However, more of the regulated proteins were found to be downregulated when compared to BSA. One could speculate that BSA may cause dramatic changes in astrocyte secretion, as astrocytes have been shown to express BSA receptors (Juurlink and Devon, 1990), rapidly endocytose the protein (Megias et al., 2000) and respond to it by stress fiber formation (Moser and Humpel, 2007). Western blotting of several of the regulated proteins in fact confirmed that BSA strongly upregulated the secretion of particularly the proteins that were downregulated by angiogenin in comparison to untreated cells. These observations could be explained by the fact that tissue culture flasks absorb many proteins, an effect that is negligible under normal culture conditions in serum-containing medium, as the plastic is saturated with serum proteins. Accordingly, by adding BSA to the otherwise serum-free medium may release plastic-bound proteins and increase their detection by mass spectrometry. Taken together, the observed regulation of protein secretion by angiogenin (containing BSA) could not be compared to the serum-free control treatment.

Visualisation of differential regulation of protein secretion in response to angiogenin treatment with the aid of BiNGO in comparison with the global gene ontology analysis performed (see Figure 39, pg. 154), revealed a shift to the regulation of proteins involved in responses to cell stimulation by angiogenin (Response to stress, Response to biotic stimulus, Response to external stimulus). However, the other annotations Metabolic process and Regulation of biological process were overall overrepresented, which 177 underlines the function of astrocytes in regulating the extracellular environment in the CNS by integrating responses to both stress and stimulation. Of the proteins regulated by angiogenin, most were in fact found to have extracellular function, suggesting that the secretion of the majority of cellular proteins was not subject to regulation by angiogenin, but may reflect basal cell activity. The most overrepresented Molecular Function, both with and without stimulation was Binding, underlining the importance of regulation of appropriate binding partners in shaping the extracellular space. Taken together, the observed shifts in astrocyte protein secretion induced by angiogenin emphasises its role in modifying astrocyte physiology to adapt to environmental changes.

Results obtained in proteomics screens need to be validated by another approach such as Western blotting and sometimes quantitative PCR. As there was a trend towards downregulation of protein secretion observed by angiogenin compared to BSA, it was particularly important to confirm SILAC results for both up- and downregulated proteins. Except for one protein tested, namely the splicing factor Srsfl, all of the proteins were found to be regulated in the same direction in Western blotting experiments as had been observed in SILAC. This could be explained by the relatively low secretion level of Srsfl compared to the other proteins tested, as Srsfl displayed the lowest intensity signal in mass spectrometry. The antibody detected intracellular Srsfl with high specificity resulting in very strong signals, thus it was concluded that the antibody was not the limiting factor in signal detection. However, the antibody detected Srsfl at 35 kDa as reported previously (Wang et al., 2001), but the signal in SILAC was found at its predicted molecular weight of 28 kDa. Accordingly, the sensitivity of the mass spectrometric approach for identifying even proteins secreted at very low levels was highlighted by this observation.

Another advantage of SILAC labelling for quantitative analysis is the fact that samples are mixed straight after harvesting. Thus, any protein loss during further sample processing would be expected to be balanced between the treatment conditions under investigation. This feature however, is lost when preparing samples for Western blotting, as the difference in mass would not be detectable by gel electrophoresis. Samples were processed in parallel, carefully avoiding subtle experimental differences which could cause inter- sample variability. Nevertheless, the exact protein regulation ratios in Western blotting showed greater variability in relation to SlLAC ratios and could therefore not be compared directly.

Taken together, the low number of proteins identified to be significantly regulated compared to the large number of total proteins detected in combination with the overall good agreement of Western blotting results for selected proteins, underline the quality of the data obtained in this screen. The BiNGO analysis further emphasises how mass spectrometric screens are useful tools to globally study protein expression and secretion changes, as they detect the most important regulatory mechanisms exerted by any stimulus and then allow to more specifically investigate the observed effects. 5. Discussion

5.1 Angiogenin lnternalisation by Astrocytes

5.1.1 The Angiogenin Receptor on Astrocytes

Based on previous studies performed to determine the role of angiogenin in ALS pathology and to gain insights into angiogenin's neuroprotective properties (Greenway et al., 2006; Kieran et al., 2008; Sebastia et al., 2009), the current study was aimed at further elucidating the functional interplay between the different cell types present in the CNS. Non-cell autonomous effects contributing to ALS pathology have long been suggested and are subject of intense investigation (reviewed by Pehar et al., 2005; and Boillee et al., 2006a; and Van Den Bosch and Robberecht, 2008; and llieva et al., 2009; and Philips and Robberecht, 201 1).

In 1997, a putative angiogenin receptor has been isolated from human endothelial cells (Hu et al., 1997), however this receptor has not been cloned or further characterised to date. In endothelial cells angiogenin translocates to the nucleus (Moroianu and Riordan, 1994a), nevertheless in angiogenin- treated mixed motoneuron cultures this was not observed by immunocytochemistry. Instead, diffuse angiogenin staining was detected in motoneurons excluding the nucleus and vesicular staining was found in surrounding glial cells. Thus, the staining pattern was very distinct from the characterised uptake into endothelial, smooth muscle and cancer cells (Moroianu and Riordan, 1994a; Li et al., 1997; Xu et al., 2001; Tsuji et al., 2005). It has long been known that angiogenin binds heparin (Soncin et al., 1997) and the current study showed that binding of heparin competes with the angiogenin receptor on astrocytes and thereby prevents its uptake. This suggested that cell surface heparansulfate proteoglycans mediated angiogenin internalisation by astrocytes. The binding of distinct proteoglycan 180 ligands can be highly specific for a certain type of heparansulfate motif like for bFGF (Esko and Selleck, 2002). The heparansulfate proteoglycan syndecan 4 is involved in bFGF signalling and internalisation (reviewed by Bass and Humphries, 2002; and Tkachenko et al., 2005; and Murakami et al., 2008; and Lambaerts et al., 2009) and was therefore investigated further. Several different experimental approaches identified syndecan 4 as bona fide angiogenin receptor on primary astrocytes. In immunocytochemistry, angiogenin colocalised with syndecan 4 in vesicles and with the aid of the in situ proximity ligation assay, it was shown that the two proteins are within a distance of less than 40 nm in these vesicles. Additionally, syndecan 4 was observed to be required for angiogenin endocytosis by astrocytes, as silencing syndecan 4 expression significantly inhibited angiogenin internalisation. Lastly, angiogenin uptake was investigated in a cell line which showed no detectable syndecan 4 expression by immunocytochemistry. These cells do not normally internalise exogenous angiogenin, but upon overexpression of syndecan 4 vesicular angiogenin staining was detected. Taken together, these findings indicate that syndecan 4 is necessary and sufficient to mediate angiogenin endocytosis by astrocytes.

Identification of syndecan 4 as the angiogenin receptor additionally provided an explanation for the observation that angiogenin was not internalised by motoneurons in vesicles: It has previously been reported that syndecan 4 was not expressed by neurons but only by glial cells in vivo (Ethell and Yamaguchi, 1999; Hsueh and Sheng, 1999), thus pointing towards signalling directionality from neurons to glia via angiogenin.

5.1.2 Angiogenin in Cell Adhesion

Cells in culture readily adhere to poly-L-Ornithine and ECM components such as fibronectin, vitronectin, collagen and fibrinogen. Similarly, angiogenin has been shown to support adhesion of endothelial and fibroblast cells (Soncin, 1992). In fact, experiments performed using the HT-29 cancer cell line indicated that very strong cell adhesion to angiogenin could be mediated by proteoglycans (Soncin et al., 1994). It has therefore been speculated, that angiogenin may itself be part of the ECM (reviewed by Strydom, 1998) and angiogenin staining was also observed in the ECM of spinal cord ventral horn biopsies (Wu et al., 2007). Angiogenin could therefore act by two different mechanisms as has been suggested for the GDNF family ligands (Bespalov et al., 201 1): These proteins exist as soluble and matrix-bound forms with distinct receptors and function. Interestingly, syndecan 3 has been identified as the receptor for the matrix-bound forms of GDNF family ligands and their interaction mediates cell spreading and neurite outgrowth. Thus, it is intriguing that syndecan 4 is the only receptor for angiogenin on astrocytes, as this may indicate that angiogenin is released by neurons to recruit astrocytes or induce cell spreading. Upregulation of angiogenin expression in neurons following focal brain ischemia has been reported in rats (Huang et al., 2009), which supports the notion that angiogenin may be released by neurons in response to stress to recruit astrocytes. Astrocytes may therefore bind angiogenin in the ECM under physiological conditions via syndecan 4, a protein well known to be required for cell attachment. However under stress, neurons could release angiogenin to increase its levels for astrocytes to endocytose free angiogenin in a syndecan 4-dependent manner triggering distinct cellular responses.

5.1.3 Angiogenin Containing Vesicles May Serve Multiple Purposes

Previous studies have shown that heparansulfate proteoglycans are involved in many endocytic and signalling events triggered by extracellular proteins, for some they serve as coreceptors, for others as primary receptors (reviewed by Belting, 2003; and Dreyfuss et al., 2009). Normally, syndecan 4 is not present on the cell surface in lipid rafts, however upon binding of ligands like bFGF it forms clusters in lipid rafts (Tkachenko and Simons, 2002; reviewed by Tkachenko et al., 2005; and Lambaerts et al., 2009). In endothelial cells, endocytosis of syndecan 4 clustered by bFGF or specific 182 antibodies triggers clathrin- and dynamin-independent internalisation via macropinocytosis (Tkachenko et al., 2004). Conversely, endocytosis of the Wnt modulator R-Spondin in a Xenopus model system was identified as clathrin-dependent process (Ohkawara et al., 201 1). Thus, the mechanism of syndecan 4 endocytosis employed probably depends on both cell type and bound ligand.

The endocytic mechanism used by astrocytes to internalise angiogenin was investigated using pharmacological inhibitors. The two most effective inhibition strategies were elevated sodium chloride and heparin treatment, which most likely inhibit initial recognition of angiogenin by its receptor syndecan 4. This was particularly evident in Western blotting, as the angiogenin signal did not decrease with the clathrin-dependent endocytosis inhibitor MDC or the dynamin inhibitor dynasore (data not shown), but sodium chloride and heparin almost completely abolished the signal (see Figure 22C and Figure 23C, pgs. 106 and 108). Thus, it could not be excluded that other endocytic mechanisms were employed by astrocytes for angiogenin internalisation. However, overexpression of the recombinant truncated clathrin adaptor AP180 or dominant-negative dynamin, led to the conclusion that angiogenin internalisation by primary astrocytes depends on clathrin for vesicle formation and dynamin for fission from the membrane.

Reports regarding the mechanism of angiogenin internalisation are incomplete (see pg. 17) and the only detail described is the lack of contribution of microtubules and lysosomes to internalisation (Li et al., 1997). In agreement with this report, its endocytosis by astrocytes was confirmed to be independent of microtubules. Accordingly, actin or intermediate filaments are probably required for endocytosis and vesicle trafficking. Since syndecan 4 serves as the receptor for angiogenin on astrocytes, its well- established cytoplasmic interaction with the actin cytoskeleton could mediate sorting via the adaptor proteins syntenin, cortactin and CASK or the actin- bundling protein a-actinin (reviewed by Simons and Horowitz, 2001; and Bass and Humphries, 2002; and Multhaupt et al., 2009).

It has previously been reported that angiogenin was internalised very rapidly and accumulated in the cell nuclei (Moroianu and Riordan, 1994a; Hu et al., 183 2000; Xu et al., 2001; Tsuji et al., 2005). However, nuclear localisation was not observed following treatment of mixed primary motoneuron cultures or primary astrocytes with angiogenin. Instead, angiogenin accumulated in vesicles, which has not been described before. Nevertheless, colabelling was only observed with some of the lysosomal vesicles stained by the markers LTR and MDC. Possibly, angiogenin is internalised primarily via clathrin- dependent endocytosis, however once the capacity is exceeded different mechanisms are employed and it is targeted for degradation as has been observed for epidermal growth factor (Sigismund et al., 2008; reviewed by Sorkin and von Zastrow, 2009). Furthermore, none of the other endosomal markers investigated displayed overlap with angiogenin staining, however distinct cell types utilise the vesicular sorting machinery very differently (reviewed by Doherty and McMahon, 2009). Thus, the identity of the majority of angiogenin-positive vesicles remains to be firmly established. Bearing in mind the integrative function of astrocytes based on the establishment of astrocytic territories (reviewed by Gordon et al., 2007; and Ben Achour and Pascual, 201 O), the observed vesicles may serve as storage compartment or could be released at a different membrane face by transcytosis. In fact, transcytosis of transferrin and BSA has been observed in astrocyte cultures (Juurlink and Devon, 1990; Megias et al., 2000) and the authors speculated that this mechanism of transporting proteins could be essential in the CNS due to the many astrocytic processes impeding diffusion of molecules in the extracellular space. Furthermore, in basal astrocyte layers, the transcytosed molecules accumulated in lysosomes after 1 h (Juurlink and Devon, 1990). This is in good agreement with the observation that some of the angiogenin was targeted to lysosomes in this study, while the majority accumulated in unidentified sorting vesicles.

Thus, it was concluded, that after binding its receptor syndecan 4, angiogenin is internalised by astrocytes in a clathrin- and dynamin-dependent manner. Some of the vesicular angiogenin is then sorted for degradation in lysosomes, while the majority of vesicles are probably transcytosed. 5.1.4 Angiogenin Escape from Vesicles

As indicated in the previous section, angiogenin internalisation has been associated with nuclear translocation as the protein contains a nucleolar targeting signal (Moroianu and Riordan, 1994b). However, its transport into the nucleus does not require the usual import mechanisms (Lixin et al., 2001). Reports about nuclear localisation of growth factors have to be interpreted with care, as both immunocytochemistry and cell fractionation approaches can lead to false identification of nuclear proteins due to technical limitations (reviewed by Olsnes et al., 2003). Nevertheless, free heparansulfate has been observed in the nuclei of a hepatoma cell line in 1986 (Fedarko and Conrad) and bFGF has been shown to accumulate in astrocytic nuclei with syndecan 2 in acute phase responses (Leadbeater et al., 2006). lmmunocytochemistry performed on astrocytes detected only very low levels of angiogenin staining in nuclei, which were more likely caused by unspecific antibody binding to nuclear components or incomplete angiogenin fixation by paraformaldehyde, which allowed for leakage of some angiogenin into the nuclei upon membrane permeabilisation. However, it cannot be excluded, that some of the angiogenin internalised in vesicles could penetrate into the cytoplasm of astrocytes to directly activate signalling or to interact with RNA or DNA. Simultaneous studies have detected RNA cleavage in mixed motoneuron cultures induced by angiogenin, which depended on angiogenin internalisation by astrocytes via syndecan 4 (M.King, J. Prehn, unpublished data). Heparansulfate proteoglycans can serve as plasma membrane carriers for Arginine-rich peptides like the DNA-binding segments of c-Fos and c-Jun (Futaki et al., 2001) and other macromolecules (reviewed by Belting, 2003). Thus the small, basic protein angiogenin could employ similar strategies to escape vesicles upon syndecan 4-dependent endocytosis to interact with RNA substrates.

The scheme below (Figure 46) shows a summary of the endocytic mechanism of angiogenin uptake by astrocytes involving syndecan 4-binding, clathrin pit formation and dynamin-dependent vesicle fission. The possible routes of internalised angiogenin are illustrated, as well as its potential involvement in ECM interactions and signalling pathways.

lntracellular

Endosome sorting ( \ for transcytosis or

Degradation? h),\ y h),\

Figure 46: Angiogenin internalisation by astrocytes. Angiogenin binds its cell surface receptor syndecan 4, which can trigger signalling via PKC, interact with the ECM or initiate endocytosis. Upon clathrin- and dynamin- dependent internalisation, angiogenin can take three possible routes: Some angiogenin containing vesicles are acidified and the contents may be degraded. The majority of vesicles may be transcytosed to signal to other cell types or serve storage purposes. Some angiogenin escapes the vesicles and triggers RNA degradation. 5.2 Angiogenin Effects on Astrocytes

5.2.1 Angiogenin Signalling

Knowledge about signalling pathways triggered by angiogenin is still incomplete and highly dependent on the respective cellular model (see pg. 18). There is evidence for involvement of both, G-protein-coupled receptors and receptor tyrosine kinases, signalling via PLC, MAP kinases or PI3 kinase. Identification of syndecan 4 as the angiogenin receptor on astrocytes indicated activation of syndecan 4-associated signalling pathways. The best studied growth factor signalling via syndecan 4 is bFGF, where low affinity binding to syndecan 4 is required for binding of its high affinity binding site on the FGF receptor (Yayon et al., 1991). However, bFGF binding can also trigger syndecan 4 signalling independently of its receptor (reviewed by Murakami et al., 2008). Syndecan 4 itself can activate PKCa or the small GTPases Racl and Rho, which in turn leads to activation of FAK (reviewed by Lambaerts et al., 2009).

Nevertheless, despite Western blotting, immunocytochemistry, CAMP measurements and ca2' imaging performed (data not shown) on angiogenin- stimulated astrocytes, it has proven difficult to exactly delineate the activated signalling pathways. The abundance of interaction partners of syndecans places them at an ideal position to collate signalling by various ligands (reviewed by Couchman, 2010), thus making the net signalling output context-dependent. Only immunostaining angiogenin-treated astrocytes for PKC displayed some colocalisation with angiogenin containing vesicles, indicating involvement of PKCa in astrocytic angiogenin signalling. PKCa is the predominant PKC isoform in astrocytes (Masliah et al., 1991). It has been reported to be involved in the mitogenic effect of insulin-like growth factor on astrocytes (Tranque et al., 1992) and to contribute to the axonal growth- promoting effect of target-derived astrocytes on spinal cord neurons (Qian et al., 1994). Hence, the first protein activated by angiogenin binding to syndecan 4 on astrocytes is most likely PKCa. However, further research 187 needs to investigate the signalling outcome focusing on the neuroprotective activities of angiogenin, especially with regard to possible implications for ALS pathology and treatment.

5.2.2 The Astrocyte Secretome in Response to Angiogenin

To date, the astrocyte secretome is poorly characterised (see pgs. 149 and 161), however many studies have pointed to the role of astrocyte- secreted proteins in neuroprotection also with regard to ALS (see pg. 11). Angiogenin has been observed to protect cultured motoneurons in paracrine (M.King, J. Prehn, unpublished data), thus it was decided to characterise changes in the astrocyte secretome in response to angiogenin stimulation.

Previous reports on modifications in the astrocyte secretome following stimulation pointed to two main regulatory pathways involved: modification of the ECM and adjustment of immune responses (see pg. 161). Interestingly, the same functional groups were regulated by cytokine (Keene et al., 2009) and cholinergic (Moore et al., 2009) stimulation, indicating that adjusting secretion of proteins belonging to these two groups may be a common mechanism by which astrocytes shape the extracellular environment. Astrocytes contribute majorly to immune responses in the CNS (reviewed by Dong and Benveniste, 2001) and glial scar formation following injury has been implicated in reduced regeneration of axons in the CNS compared to the peripheral nervous system (reviewed by Yiu and He, 2006). Inflammatory responses of astrocytes are also associated with ALS (see pg. 1 I), which further highlights the importance of appropriate astrocyte function also with regard to neurodegeneration.

In accordance with previously published data, the current study identified many components of the ECM to be regulated by angiogenin, such as osteopontin, neurocan, olfactomedin-like protein 3, thrombospondin 2 and collagen IV, and enzymes modifying the composition of the ECM by their enzymatic activity like MMP3, MMPl9, Plau, plasminogen activator inhibitor 1, alpha-2-macroglobulin and metalloproteinase inhibitor I. Furthermore, components of the complement system and the cytokines interleukin-6 and -12 were regulated by angiogenin. Thus, context-dependent fine tuning of astrocytic responses to their environment has to be taken into account in more detail, as global changes allow for only limited conclusions to be drawn with regard to specific regulatory mechanisms based on the data obtained.

When taking the specific role of angiogenin in angiogenesis (see pg. 25) and previous publications into account, it is interesting to observe regulation of proteins involved in plasmin activity by angiogenin stimulation of astrocytes: Angiogenin upregulated the secretion of the plasminogen activator Plau, while it downregulated the plasminogen activator inhibitor. However, it has been reported that Plau is downregulated and the plasminogen activator inhibitor upregulated following cholinergic stimulation and the authors claim this might aid neuritogenesis (Moore et al., 2009). The role of the plasmin system in the CNS especially with regard to consequences on neuronal behaviour still remains to be elucidated: Plau has been reported to promote recovery of motoneurons following spinal cord injury by signalling via its receptor (Seeds et al., 201 I). However, the plasminogen activator inhibitor I has been observed to protect neurons from apoptosis (Soeda et al., 2008) and treating the SOD1 ALS mouse model with a Plau inhibitor prolonged survival and improved motor performance (Glas et al., 2007). Interpretation of results is further complicated by the fact that Plau can act as plasminogen activator only after proteolytic activation or by binding its cell surface receptor, which triggers intracellular signalling (reviewed by Myohanen and Vaheri, 2004). Accordingly, the observed regulation of the plasmin system by angiogenin needs to be further investigated, especially with regard to possible microenvironmental changes in ALS that could be positively affected by upregulating Plau secretion. 5.2.3 Fine Tuning of Astrocyte Secretion

Several astrocyte-secreted proteins regulated by angiogenin have been selected for validation by Western blotting based on the observed SlLAC ratios and published data indicating involvement in neuronal survival (indicated in bold letters; see pg. 174). The role of Plau in CNS physiology has been discussed in the previous section, however plasminogen activators and MMPs often act in concert and plasmin can activate MMP3 (reviewed by Myohanen and Vaheri, 2004). Stress-induced activation of intracellular MMP3 has been observed in neurons triggering apoptosis (Choi et al., 2008; Kim et al., 2010) and release of active MMP3 by neurons has been implicated in microglial activation and blood brain barrier breakdown (reviewed by Kim and Hwang, 201 1). Thus, the functional role of MMP3 in neurodegeneration could be related to removal of neurons that are beyond repair. Secretion of the inactive pro-form of MMP3 by astrocytes was downregulated by angiogenin and low levels of active, intracellular 48 kDa MMP3 were detected by Western blotting (see Figure 45; pg. 176). This indicated that angiogenin may protect motoneurons by attenuating immune responses and blood brain barrier degradation, which is observed in ALS (Garbuzova-Davis et al., 2007a; Garbuzova-Davis et al., 2007b; Zhong et al., 2008; Nicaise et al., 2009; reviewed by Garbuzova-Davis et al., 201 1).

Breakdown of the blood brain barrier requires degradation of the basal lamina surrounding cerebral blood vessels, which consists of ECM components such as laminin, fibronectin and Col IV that are substrates of MMP3 (reviewed by Kim and Hwang, 201 1). Col IV isoforms are involved in formation, maturation and maintenance of neuromuscular junctions (Fox et al., 2007). However, the effect of Col IV deposition on axonal regrowth after injury is controversial (Stichel et al., 1999; Joosten et al., 2000; Hubert et al., 2009). These results could be explained by different injury models, as local differences in scar formation in the CNS have been reported (Alonso and Privat, 1993) and responses to injury in the spinal cord may be more vigorous (Schnell et al., 1999). Nevertheless, upregulation of Col IV secretion by cultured astrocytes has been observed in response to stimulation with 190 cytokines like TGF-PI, interferon-y or interleukin-1P (Liesi and Kauppila, 2002). The observed downregulation of Col lV expression in response to angiogenin could therefore counteract negative effects of CNS inflammation in ALS.

The ECM protein Tgfbi was originally detected to be secreted by human adenocarcinoma cells in response to TGF-PI stimulation (Skonier et al., 1992) and inhibits in vitro cell attachment (Skonier et al., 1994). Interestingly, the protein binds Col IV amongst other collagens (Hashimoto et al., 1997) and has been reported to promote in vitro cell adhesion of a human astrocytoma cell line (Kim et al., 2003). TGF-P isoforms have been shown to regulate motoneuron survival (reviewed by McLennan and Koishi, 2002) and have been discussed as target for ALS therapy (Day et al., 2005; reviewed by Katsuno et al., 201 1). However, upregulation of TGF-PI has only been observed at late disease stages in patient CSF and plasma (Houi et al., 2002; llzecka et al., 2002). Taken together, angiogenin-upregulated secretion of Tgfbi could at least partially mimic the observed protective effects of TGF-P isoforms by modulating cell adhesion to the ECM.

Treatment of SODl mice with ILlra, the inhibitor of interleukin-I receptor binding, extended the life span of mice and reduced neuroinflammation (Meissner et al., 2010), thus blocking interleukin-IP has been suggested as therapeutic approach for ALS (reviewed by van der Meer and Simon, 2010). Similarly, SODl mice overexpressing a dominant-negative mutant of the interleukin-lP-converting enzyme displayed slowed progression of ALS pathology (Friedlander et al., 1997), however knocking down interleukin-I p in SODl model mice did not alter disease course (Nguyen et al., 2001). Accordingly, it was very surprising to observe downregulation of lLlra secretion by astrocytes in response to angiogenin. Nevertheless, in primary astrocyte cultures interleukin-I p has been reported to upregulate the uptake of L-Glutamate together with prostaglandin E2 (Okada et al., 2005). Therefore, angiogenin may locally modulate astrocytic Glutamate clearance and prevent excitotoxicity by increasing interleukin-I p activity.

Histone H4 has previously been detected in astrocyte-conditioned medium (Greco et al., 2010), despite lacking a signal peptide or otherwise sharing 191 homology with unconventionally secreted proteins. Lately, secreted histones have attracted attention as part of so-called NETS (Neutrophil extracellular traps), which additionally contain decondensed chromatin and anti-microbial proteins (reviewed by Papayannopoulos and Zychlinsky, 2009). Like other antimicrobials, histones can lyse eukaryotic cells, however they may also serve as intra- and extracellular lipopolysaccharide sensors (Augusto et al., 2003). On the surface of cerebellar neurons in culture extracellular histone HI has been observed, where it stimulated neuritogenesis and process formation (Mishra et al., 2010). Thus, the exact role of histone H4 secretion by astrocytes remains to be elucidated, however its downregulated secretion in response to angiogenin indicated it may be released as part of the astrocytic immune response and reduce toxicity to neurons.

The current screen identified the secretion of five members of the SR protein family of splicing factors to be downregulated by angiogenin treatment of astrocytes, namely Srsfl, Srsf2, Srsf3, Srsn and SrsflO. As discussed before (see pg. 177), confirmation of the observed downregulation was impossible due to low secretion levels, however extracellular localisation of splicing factors has been reported in secreted vesicles (Lee et al., 2009) and on the surface of endothelial cells (Hatakeyama et al., 2009). As cultured astrocytes have been observed to secrete exosomes (Faure et al., 2006), it seems possible that these may also contain splicing factors. Both proteins and RNAs have been discussed to be transferred between neurons and possibly astrocytes at synapses via exosomes (reviewed by Smalheiser, 2007), and glial supply of mRNA for neurons may be correlated with the size of neuronal cells (reviewed by Giuditta et al., 2008). Interestingly, some SR family proteins may be involved in mRNA export from the nucleus and Srsfl regulates cap-dependent translation (Karni et al., 2007; Michlewski et al., 2008; reviewed by Shepard and Hertel, 2009). In conclusion, astrocytes may locally regulate mRNA transport and translation in neurons in response to angiogenin treatment, which possibly helps the neuron adapt to stress via reducing cap-dependent translation. 5.2.4 Angiogenin in the Brain: Signalling Directionality

The results obtained in the present study shed new light on the functional interplay between neurons, astrocytes, microglia, endothelial cells and the surrounding ECM. This complex regulatory network is markedly influenced by angiogenin levels in the CNS.

Basal angiogenin levels in the brain are high (Moreau et al., 2009), therefore angiogenin might constitute part of the ECM and contribute to cell adhesion of non-neuronal cells via its binding to syndecan 4 on the cell surface. Motoneurons basally express angiogenin (Greenway et al., 2006; Wu et al., 2007; Kieran et al., 2008), however upon stress neuronal expression of angiogenin increases further (Huang et al., 2009). The angiogenin released by neurons is then taken up by astrocytes in close proximity via syndecan 4, a process further controlled by local extracellular ion concentration depending on neuronal activity (reviewed by Kahle et al., 2009) and upregulation of syndecan 4 expression in reactive astrocytes (Iseki et al., 2002).

Some of the internalised angiogenin escapes endosomes and alters astrocytic RNA metabolism. Alternatively, astrocytes endocytose angiogenin secreted by neurons and may pass the protein on to other cell types such as endothelial cells (Figure 47), as angiogenin has been shown to enhance proliferation of endothelial cells (Kishimoto et al., 2005). Syndecan 4 expression has been reported on endothelial cells, thus angiogenin may stimulate endothelial proliferation via PKCa like bFGF (Murakami et al., 2002) and NO production by endothelial cells leading to increased arterial blood flow (Partovian et al., 2005). Thirdly, angiogenin binding to syndecan 4 and subsequent endocytosis triggers signalling within the astrocytes. This leads to changes in the proteins secreted by astrocytes, which affects all surrounding cell types. Astrocytes functionally organise brain architecture into territories establishing so-called glio-vascular units, in which astrocytes contact neurons and endothelial cells (reviewed by Nedergaard et al., 2003). This places astrocytes at an ideal position for regulating brain homeostasis

193 locally and globally via the cerebrovasculature (reviewed by Attwell et al., 201 0).

Mass spectrometry analysis of the astrocyte secretome in response to angiogenin further illustrates the importance of local protein secretion in regulating functional cellular networks: Astrocytes majorly shape the ECM by secreting ECM components and by modifying existing structures like the basal lamina via protease release. The secreted proteases also activate microglia, as do proteins classically associated with immune responses like complement components, histones and other immunomodulators. Microglia similarly secrete neuroprotective (or neurotoxic) factors, thus affecting motoneuron survival. Immune proteins may also have autocrine effects on astrocytes themselves. Astrocytes more actively aid neurons by transferring RNAs and proteins (reviewed by Smalheiser, 2007; and Giuditta et al., 2008), and the screen identified splicing factors and ribosomal proteins to be secreted by astrocytes presumably via exosomal vesicles.

In conclusion, angiogenin modifies the secretory profile of astrocytes in a way that may locally aid motoneuron survival by supplying components required for local protein translation, which may be of particular importance with regard to the size of individual motoneurons. Astrocytes also positively influence the local environment of motoneurons in response to angiogenin by supporting endothelial cells in maintaining the basal lamina and regulating the blood flow. Furthermore, microglial responses to inflammatory stimuli are adjusted by astrocytic responses to angiogenin. In conclusion, our understanding of the exact regulatory mechanisms and all the interaction partners involved is still rudimentary, however the results of this study reveal astrocytes as crucial players in motoneuron protection by angiogenin. Figure 47: Summary of astrocytic responses to angiogenin stimulation. Proteins secreted by astrocytes greatly influence the surrounding cells either directly or by modifying the ECM: Proteases act on the basal lamina, the ECM or activate microglia; ECM components affect scar formation and cell adhesion; Proteins involved in immune responses (complement factors, histones, immunomodulators) act on microglia and astrocytes themselves; Components of the plasmin system signal to neurons; Splicing factors and ribosomal proteins change neuronal protein translation. Angiogenin secreted by neurons is endocytosed by astrocytes and adjusts secretion of proteins locally modifying the microenvironment to support motoneurons. Additionally, angiogenin may be passed on to endothelial cells to modulate the cerebrovasculature. 5.3 Future Perspectives for ALS Research

Despite the fact that ALS has first been described over 140 years ago, the only disease-modifying drug currently available to treat ALS is still the inhibitor of glutamate release Riluzole. Otherwise, treatments alleviating symptoms and thereby improving quality of life are commonly part of ALS patient management. The search for effective treatment strategies is hampered by the heterogeneity in disease phenotype and inadequate clinical trials. The majority of preclinical trials have been performed using the SODl mouse model, which only allows for limited conclusions to be drawn regarding effects on ALS patients. Additionally, preclinical trials often start treatment before symptom onset and therefore primarily aid to elucidate mechanisms contributing to motoneuron degeneration, rather than developing new treatment strategies (reviewed by Kiernan et al., 201 I).

The current study was aimed at gaining further insights into the physiological role of angiogenin in the CNS and its contribution to the pathomechanims involved in ALS, based on the fact that angiogenin mutants have been associated with ALS patients (Greenway et al., 2006) and the wildtype protein displays potent neuroprotective properties (Kieran et al., 2008; Sebastia et al., 2009). However, the identification of syndecan 4 as the receptor for neuron-derived angiogenin on astrocytes raises many new questions: Besides the established upregulation of angiogenin in motoneurons in response to hypoxia (Sebastia et al., 2009), the physiological trigger for secretion of angiogenin by motoneurons remains unknown. One could also speculate that following breakdown of the blood spinal cord barrier additional plasma angiogenin may flood the CNS. Besides changes in angiogenin levels, syndecan 4 is subject to regulation not only in reactive astrocytes (Iseki et al., 2002), but interestingly strong upregulation of syndecan 4 has been reported in late stage SODl mutant motoneurons (Ferraiuolo et al., 2007). Thus, angiogenin and syndecan 4 levels in the central nervous system are tightly regulated to trigger appropriate responses by astrocytes and other cell types in the CNS. Astrocytes respond to angiogenin in multiple ways to integrate a plethora of signalling inputs. They majorly contribute to the structure of the ECM, support neurons by supplying growth factors and proteins required for cellular and metabolic processes and modulate immune responses. Accordingly, subtle changes in the physiological function of angiogenin due to mutations may be sufficient to tilt the proper balance in the brain and lead to damage to neuronal cells. However, the exact role of angiogenin mutations will need to be elucidated further especially with regard to signalling capacity between cells, bearing in mind the possible routes internalised angiogenin may take.

The identification of the angiogenin receptor on astrocytes and their response to angiogenin stimulation with regard to uptake and protein secretion has extended current knowledge about the functional interplay between cells in the CNS. However, these results need to be further investigated focusing on their implications for the pathomechanism involved in ALS to develop more targeted therapies in the future. 6. Bibliography

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