The signalling and function of EB12 in the central nervous system

LEABHARLANN CHOLAISTE NA TRIONOIDE, BAILE ATHA CLIATH TRINITY COLLEGE LIBRARY DUBLIN

OUscoil Atha Cliath

The University of Dublin

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The Signalling and Function of EBI2 in the Central

Nervous System

This thesis submitted to University of Dublin, Trinity College

for the degree of

Doctor of Philosophy

Aleksandra Rutkowska

O cto ber 201 4)

Supervisor:

Prof. Kumlesh K. Dev

Molecular Neuropharmacology,

D epartm ent of Physiology, School o f Medicine,

D ec la ratio n and s ta te m e n t o f plagi.irism

I declare that this thesis has not been submitted as an exercise for a degree a t this or any other university and it is entirely my own work.

I agree to deposit this thesis in the University's open access institutional repository or allow the library to do so on my behalf, subject to Irish Copyright Legislation and Trinity College Library conditions of use and acknowledgement.

Signed Date

TRWITY COLLEGE

LIBRARY DUBLIN

Acknowledgments

I am grateful to my supervisors, Prof. Kumlesh Dev and Dr. Andreas Sailer fo r th e ir continuous support and much valued advice and guidance. I am particularly thankful to Dr. Sailer fo r giving me

the opportun ity to spend the last three summers in Novartis. Thanks to his easy-going nature and passion fo r science he made my tim e spent in Novartis very productive as w/ell as enjoyable. I have learned a great deal o f techniques and had a unique opportunity to experience how science is done

in a big pharma.

M y gratitude also goes to prof. Dev fo r his ongoing guidance and support in solving everyday problems encountered in the lab. I am also grateful fo r his innovative ideas and enthusiasm w/hen

em barking new/ concepts. I am particularly thankful fo r giving me the opportun ity to contribute to several publications and also fo r the tim e spent teaching me how to w rite papers.

I would also like to thank the current and form er lab members who provided advice and experim ental support: Dr. Adam Pritchard, Sinead O'Sullivan, Cat O'Sullivan, Dr. Graham Sheridan, Dr. Debadutta Deb, Dr. Marika Doucet and Dr. Luke Healy. My thanks also go to colleagues in

T a b le o f c o n te n ts

D e c la ra tio n a n d s ta t e m e n t o f p la g ia r is m ...ii

A c k n o w le d g m e n ts ... iii

T a b le o f c o n te n ts ... iv

List o f fig u re s a n d t a b le s ... v iii T a b le o f c o m m o n a b b r e v ia tio n s ... x

S c ie n tific a b s t r a c t ...xi

Lay a b s tr a c t... x ii A im s a n d h y p o th e s is ... x iii V a lu e o f r e s e a r c h ... x iv O u tp u ts ...XV C h a p te r 1: In t r o d u c t io n ...1 7 O v e rv ie w ... 18

1 EBI2 Receptor...20

1.1 Structure and expression of EBI2... 20

1.2 Signalling o f EBI2... 20

1.3 Oxysterols...21

1.4 EBI2 function in th e im m une sy s te m ... 22

2 A stro cytes...30

2.1 Astrocyte origins and typ es... 30

2.2 Astrocyte fu n ctio n s...30

2.3 Similarities and differences b etw een rodent and human astrocytes... 31

2.4 Astrocyte involvem ent in disease... 31

3 Oligodendrocytes... 37

3.1 Oligodendrocyte m orphology and fu nction... 37

3.2 Myelin developm ent and m aintenan ce... 37

3.3 Myelin-specific and o th er proteins expressed in oligodendrocytes...38

3.4 Diseases o f o ligo d en drocytes... 39

4 EBI2 and oxysterols in disease... 43

4.1 EBI2 in disease... 43

4.2 Hereditary spastic paraplegias type 5 ...43

4.3 Alzheimer's disease... 43

4 .5 Parkinson's disease...45

4 .6 A ge-related m acular d e g e n e ra tio n ... 45

5 Closing s ta te m e n t... 47

C h a p te r 2 : M a t e r ia ls a n d m e t h o d s ...4 8 1 M a te r ia ls ... 49

1.1 C o m po u nd s... 49

1.2 A n tib o d ie s ... 49

1.3 S tains... 50

2 Anim al h usb an d ry... 50

3 Cell C u ltu re ...50

3.1 Hum an astrocyte c u ltu re ...50

3.2 M u rin e astrocyte c u ltu re ...50

3 3 Culture o f mouse organotypic slices... 51

3.4 H eterologous cell line c u ltu re ... 51

4 M o lecu lar B iology... 52

4 .1 Preparation o f m R N A ...52

4.2 Real tim e q u an titative polymerase chain reaction (RT-qPCR)... 52

5 B io ch em istry... 52

5.1 Proteom e profiler antibody a rra y s ... 52

5.2 Hom ogeneous tim e resolved fluorescence (HTRF)... 53

5.3 The enzym e-linked im m unosorbent assays (ELISAs)...53

5.4 W estern blot analysis...54

6 Cellular staining s tu d ies... 55

6.1 Fluorescence-activated cell sorting (FACS)...55

6.2 Im m u no cyto ch em istry... 55

6.3 M TT assay... 56

7 Functional Studies... 56

7.1 Calcium signalling... 56

7.2 xCELLigence im pedance assay...56

7.3 M igration and w/ound healing assays...57

8 Statistical analysis... 57

C h a p te r 3: EBI2 re g u la te s in tr a c e llu la r s ig n a llin g a n d m ig r a tio n in a s tr o c y t e s ...6 1 C hapter a im s ... 62

1 Introduction 64

2 Results... 66

2.1 EBI2 Is expressed in human and mouse astrocytes... 66

2.2 Human and mouse astrocytes express enzymes necessary fo r 7a25H C synthesis and d egradatio n ... 66

2.3 EBI2 activation induces pERK 1 /2 signalling in human astrocytes... 66

2 .4 Agonism of EBI2 induces Ca^^ signalling in human astrocytes...67

2.5 EBI2 regulates wound healing/m igration in human astrocytes... 67

2 .6 7a25H C does not induce migration in EBI2 knock-out a s tro c y te s... 68

3 Discussion... 75

3.1 Astrocytes are involved in norm al and pathophysiological processes in th e CNS...75

3.2 Astrocytes express EBI2 and 7a25H C synthesising and degrading e n z y m e s ... 75

3.3 EBI2 induces m igration/w ound healing in astrocytes... 76

C h a p te r 4: EBI2 re g u la te s lev e ls o f IL6 a n d T N F a in a s tro c y te s a n d t h e ir s ig n a llin g w ith m a c r o p h a g e s ... 7 7 Chapter a im s...78

A bstract... 79

1 In tro d u c tio n ...80

2 Results... 82

2.1 LPS does not induce cytokine release in human astrocytes... 82

2.2 7a25H C attenuates IL17/TNFa-induced levels of IL6 and NFkB translocation to th e nucleus in human astrocytes...82

2.3 EBI2 agonism inhibits IL17/TN Fa signalling in human astrocytes...83

2.4 7a25H C attenuates LPS-induced levels o f IL6 and TNFa in rodent astrocytes...83

2.5 LPS induces changes in mRNA levels of EBI2 and 7a25H C related enzymes in mouse astrocytes... 83

2.6 Mouse astrocyte conditioned m edia induces macrophage (R AW 264.7) m igration...84

3 Discussion... 91

3.1 EBI2 Inhibits pro-inflam m atory cytokine release and NFkB signalling in astrocytes...91

3.2 EBI2 regulates inter-cellular com m unication betw een astrocytes and m acrophages... 92

C h a p te r 5: EB I2 re g u la te s m y e lin d e v e lo p m e n t a n d in h ib its L P C -in d u c e d d e m y e lin a tio n . 9 3 C hapter a im s ...94

A bstract... 95

1 In tro d u c tio n ...96

2.1 Organotypic cerebellar slices maintain host tissue cytoarchitecture and physiology...99

2.2 EBI2 deficiency results in transient delay in MBP expression... 99

2.3 O lig l, CNPase and MOG are not differentially expressed in EBI2 KO and WT m ic e ...100

2.4 Downregulation o r antagonism o f EBI2 signalling staggers m ye lin a tio n ... 100

2.5 7a25HC attenuates LPC-induced demyelination in mouse cerebellar slices...100

2.6 EBI2 attenuates LPC-induced IL6 and IL1(3 release in mouse cerebellar slices... 101

3 Discussion... 109

3.1 EBI2 is involved in myelin develop m e nt... 109

3.2 Inhibition o f EBI2 signalling leads to delay in MBP expression... 110

3.3 EBI2 protects from LPC-induced demyelination via inhibition o f pro-inflam m atory cytokine release...110

Chapter 6: Discussion... 112

6.1 Study overview/...113

6.2 The role of astrocytes in CNS physiology... 113

6.3 Expression of EBI2 and 7a25HC synthesising and degrading enzymes in astrocyte biology 114 6.4 The role of EBI2 in astrocyte m ig ra tio n ...114

6.5 Regulatory role o f EBI2 and oxysterols in in fla m m a tio n ...117

6.6 The role of EBI2 in inflam m atory response in astrocytes... 117

6.7 The role of EBI2 in astrocyte-mediated cellular cross-talk... 117

6.8 EBI2 signalling in myelin developm ent... 120

6.9 The role of EBI2 in protection from chemically induced dem yelination... 120

6.10 Limitations and future directions... 123

6.11 Closing remarks... 123

List of figures and tables

C hapter 1: In troduction

Figure 1.1 Hum an, mouse, and rat EBI2 sequences 24

Figure 1.2 EBI2 phylogenetic tree 25

Figure 1.3 EBI2 expression in cells and tissue 26

Figure 1.4 EBI2 signalling pathways 27

Figure 1.5 Chemical structure and synthetic pathways of cholesterol, related oxysterols and

EBI2 antagonists

28

Figure 1.6 Function of EBI2 in th e im m une system 29

Table 1.1 Astrocyte functions 33

Figure 1.7 Receptors and ligands expressed in astrocytes 34

Table 1.2 Differences betw een human and ro d ent astrocytes 35

Figure 1.8 Cellular processes involved in m ultiple sclerosis 36

Figure 1.9 O ligodendrocyte markers expressed during developm ent 41

Table 1.3 M yelin-specific proteins and th e ir function 42

Table 1.4 EBI2 and oxysterols implicated in diseases 46

C hapter 2: M a te ria l and M etho d s

Table 2.1 List of prim ary and secondary antibodies 58

Figure 2.1 Experim ental setup using EClS and xCELLigence platforms 59

C hapter 3: EBI2 regulates intracellular signalling and m igratio n in hum an astrocytes

Figure 3.1 EBI2 is expressed in human and m ouse astrocytes 68

Figure 3.2 Human and mouse astrocytes express enzymes necessary fo r 7a25H C synthesis 69

and degradation

Figure 3.3 EBI2 activation induces ERK signalling in hum an astrocytes 70

Figure 3.4 EBI2 activation induces Ca^* signalling in hum an astrocytes 71

Figure 3.5 7a25H C induces human astrocyte w ound healing/m igration 72

Figure 3.6 7a25H C does not induces m igration in EBI2 knock-out astrocytes 73

C hapter 4: EBI2 regulates levels o f IL6 and T N F a in astrocytes and th e ir signalling w ith

___________ macrophages_____________________________________________________________

Figure 4.1 IPS does not induce release o f cytokines and chemokines in human astrocytes 84

Figure 4.2 7a25H C attenuates IL17/TN Fa-induced levels of IL6 and NFkB nuclear 85

translocation in human astrocytes

Figure 4.3 7 a25H C inhibits IL17/TN Fa-induced signalling in human astrocytes 86

Figure 4 .4 7 a25H C attenuates LPS-induced levels o f IL6 and TNFa in rodent astrocytes 87

Figure 4.5 LPS induces changes in mRNA levels o f EBI2 and 7 a25H C synthesizing and 88

degrading enzymes in mouse astrocytes

Figure 4 .6 M ouse astrocyte conditioned m edia induces m acrophage (R AW 264.7) migration 89

C hapter 5: EBI2 regulates m yelin d e v e lo p m e n t and inhibits LPC-induced d e m y e lin a tio n

Figure 5.1 Organotypic cerebellar slices m aintain host tissue cytoarchitecture and physiology 102

Figure 5.2 EBI2 deficiency results in transient delay in MBP expression 103

Figure 5.3 O lig l, CNPase and MO G are not differentially expressed in W T and EBI2 KO mice 104

Figure 5.5 7 a25H C protects from LPC-induced dem yelination

Figure 5 .6 7 a25H C attenuates LPC-induced levels o f IL ip and IL6 in cerebellar slices

106 107

C hapter 6: Discussion

Figure 6.1 EBI2 regulates intracellular signalling and m igration in astrocytes 115 Figure 6.2 EBI2 regulates levels o f IL6 and TNFa in astrocytes and th e ir signalling w ith 118

macrophages

T ab le o f c o m m o n a b b re viatio n s

Abbreviation Definition Abbreviation Definition

7a25HC

la, 25-dihydroxycholesterol

Hypoxanthine-guanine

phosphoribosyltransferase

Ab

Antibody

Cholest-5-ene-3P,7a-diol 3(3-

dehydrogenase

AD

Alzheimer disease

Interferon

APC

Antigen presenting cell

Interleukin

BBB

Blood brain barrier

Knock-out

BCR

B cell receptor

Lysophosphatidylcholine

BDNF

Brain derived neurotropic factor

Lipopolysaccharide

BSA

Bovine serum albumin

Liver X receptor

CCL/CXCL/...

Chemokine ligand

Mitogen-activated protein

CCR/CXCR/...

Chemokine receptors

Myelin basic protein

CH25H

Cholesterol-25-hydroxylase

Myelin oligodendrocyte

glycoprotein

CNS

Central nervous system

Marginal reticular cell

CHO

Chinese hamster ovary cell

Multiple sclerosis

Cl

Cell index

3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide

CSF

Cerebrospinal fluid

Nuclear factor of activated I

cells

CYP7B1

Hydroxycholesterol 7-alpha-

hydroxylase

NFkB

Nuclear factor kappa B

DC

Dendritic cell

Natural killer cell

DMSO

Dimethyl sulfoxide

Nitric oxide

EAE

Experimental autoimmune

encephalomyelitis

OPC

Oligodendrocyte progenitor cell

EBI2

Epstein-Barr virus-induced gene

Parkinson's disease

EBV

Z

Epstein-Barr virus

Phosphate buffered saline

EClS

Electric Cell-Substrate

Impedance Sensing (EClS)

PBMC

Peripheral blood mononuclear

cell

ERK

Extracellular-signal -regulated

kinase

PLP

Proteolipid protein

FACS

Fluorescence-activated cell

sorter

Ptx

Pertussis toxin

FBS

Fetal bovine serum

Reactive oxygen species

FDC

Follicular dendritic cell

Sphingosine-l-phosphate

FGF

Fibroblast growth factor

SIP receptor 1

GC

Germinal centre

Tris-buffered saline

GFAP

Glial fibrillary acidic protein

Toll-like receptor

GPCR

G-protein coupled receptor

Tumor necrosis factor alpha

Scientific abstract

T h e G p ro te in -c o u p le d re c e p to r EBI2 (E p s te in -B a rr viru s -in d u c e d g e n e 2) is a c tiv a te d by 7 a , 2 5 - d ih y d ro x y c h o le s te ro ! (7 a 2 5 H C ) an d plays a ro le in T c e ll-d e p e n d e n t a n tib o d y resp on se an d B cell m ig ra tio n . A b e r ra n t EBI2 signalling is im p lic a te d in a ra n g e o f a u to im m u n e disorders h o w e v e r, its

ro le in th e CNS re m a in s u n k n o w n . H e re w e c h a ra c te rize th e fu n c tio n a l ro le o f EBI2 in th e brain using p rim a ry h u m a n an d m o u se astro cy tes, o rg a n o ty p ic c e re b e lla r slice cu ltu res and EBI2 kn o ck o u t an im als. W e fin d th a t h u m a n an d m o u se astro cytes express EBI2 and th e en zym es necessary fo r synthesis an d d e g ra d a tio n o f 7 a 2 5 H C . In astro cy tes, EBI2 a c tiv a tio n s tim u la te s ERK p h o s p h o ry la tio n ,

sig nalling an d induces c e llu la r m ig ra tio n (R esu lts 1), in a d d itio n to a tte n u a tin g LPS an d IL 1 7 /T N F a -m e d ia te d re lease o f IL6 an d T N F a in h u m an and m o u se astro cytes. A c tiv a tio n o f EBI2 also inhibits IL 1 7 /T N F a -in d u c e d signalling an d N FkB tra n s lo c a tio n to th e nucleus. Im p o rta n tly , analysis o f

m R N A levels o f EBI2 an d its signalling p a th w a y in m o u se astro cy tes tr e a te d w ith LPS s h o w e d th a t th e s e cells re s p o n d to im m u n e c h allen g e in p a rt by re g u la tin g th e expression p a tte rn o f EBI2 an d

o xys tero l re la te d genes. T h e d a ta also s h o w e d th a t as tro cy tes s tim u la te d w ith LPS can in d u ce m a c ro p h a g e m ig ra tio n and th a t EBI2 can re g u la te th is c e llu la r crosstalk (R esu lts 2 ). In a d d itio n to

playing im p o r ta n t roles in a s tro c y te b iology and im m u n e system re g u la tio n in th e CNS, th e d a ta s h o w e d t h a t EBI2 signalling is involved in m y elin d e v e lo p m e n t and th a t in h ib itio n o f EBI2 signalling induces a d e la y in m y elin d e v e lo p m e n t. W e also s h o w th a t EBI2 signalling is p ro te c tiv e in LPC- ind u ced d e m y e lin a tio n in o rg a n o ty p ic c e re b e lla r slice c u ltu re s and th a t th e p ro te c tio n is, a t least

p artia lly , m e d ia te d via in h ib itio n o f p ro -in fla m m a to ry c y to k in e re lease (R esu lts 3 ). These results, fo r th e first tim e , d e m o n s tra te a ro le fo r EBI2 in a s tro c y te fu n c tio n and m yelin d e v e lo p m e n t and p ro te c tio n ag ain st d e m y e lin a tin g insults and suggest t h a t m o d u la tio n o f th is re c e p to r m a y be

Lay a bstract

The four major brain-specific cell types in the brain are; (i) neurons, which transm it and process

inform ation via electrical signals, (ii) oligodendrocytes, which insulate neuronal axons w ith fatty

sheets called myelin to ensure fast and complete electrical signal transduction, (iii) microglia, which

provide defence against potential infections in the brain and, (iv) astrocytes, th a t serve many

im portant roles such as immune defence, cleaning up debris, ensuring proper neuronal functioning

and also communication w ith other cells. These cells, and indeed all living cells, have a wide range of

proteins called receptors on th e ir surface, which are activated by signalling molecules. EBI2 is one

such receptor that plays very im portant roles in the biology of immune cells, where it regulates

antibody production to fight infections. In this thesis, we investigated w hether EBI2 is also present

on the cell surface of brain cells, namely astrocytes. We asked w hether EBI2 receptor is involved in

regulation of astrocyte physiology and fighting o f infections in the brain, and also if it can regulate

oligodendrocyte myelination o f neurons. Our research has shown fo r the first tim e that EBI2 is

present on the surface o f astrocytes and that it does play a role in immune function in the brain.

When EBI2 is in contact w ith its signalling molecule (called oxysterol 7a25HC) it causes the

astrocytes to migrate and also stops these cells from releasing harmful inflam m atory signals. The

EBI2 receptor is also capable of sensing infections and responding to them by releasing molecules

that attract immune cells into the CNS, to help fight infection. In addition, we found that the EBI2

receptor is necessary fo r appropriate and tim ely myelination of neurons and that activation o f the

EBI2 receptor can protect the cells from chemically induced loss of myelin. These findings are

im portant because they inform us about the role of the EBI2 receptor in the brain, and about how

astrocytes and oligodendrocytes are modulated by this receptor. These findings also allow us to

develop new ways to control brain cell function and lim it pro-inflam m atory processes. We believe this w ork is im portant fo r development of new drugs that might be beneficial in treating diseases

A im s an d hypothesis

The aims o f th e study are as fo llo w s:

• To investigate EBI2 expression in th e cells o f th e central nervous system, specifically

astrocytes (Results 1).

• To id e n tify w h e th e r astrocytes express enzymes necessary fo r th e EBI2 ligand, oxysterol

7a25HC, synthesis and degradation (Results 1).

• To e xplore EBI2 signalling pathw ays in astrocytes (Results 1).

• To study w h e th e r EBI2 agonism induces astrocyte m igration (Results 1).

• To exam ine if EBI2 in h ibits p ro -in fla m m a to ry cytokine release (Results 2).

• To exam ine w h e th e r activation o f EBI2 attenuates p ro -in fla m m a to ry signalling in astrocytes

(Results 2).

• To investigate w h e th e r EBI2 and 7a25HC related enzymes are regulated in response to

im m une challenge (Results 2).

• To e xplore the role o f EBI2 in astrocytes in cellular cross-talk (Results 2).

• To study th e role o f EBI2 signalling in m yelin d eve lo pm e nt in vivo and in vitro using w ild typ e and EBI2 knock-out mice (Results 3).

• To id e n tify th e role o f EBI2 signalling in m yelin deve lo pm e nt (Results 3).

• To investigate w h e th e r EBI2 protects fro m chem ically-induced d em yelination (Results 3).

• To exam ine th e role o f EBI2 in p ro -in fla m m a to ry cytokine signalling during chem ically-

induced dem yelina tion (Results 3).

W e hypothesise th a t EBI2 is expressed in astrocytes and th a t it regulates astrocyte signalling and

fu n ctio n . Specifically, we hypothesise th a t astrocytes express EBI2 and enzymes necessary fo r

7a25HC synthesis and degradation, EBI2 agonism w ith 7a25HC activates dow nstream signalling

pathways, EBI2 stim ulates m igration o f astrocytes, EBI2 alters cyto kine /che m okine levels in

astrocytes, EBI2 and 7a25H C -related enzymes are regulated in astrocytes upon im m une challenge,

and th a t EBI2 in astrocytes is involved in in te r cellular com m unication. We also hypothesise th a t EBI2

is involved in m yelin d evelopm ent, th a t EBI2 a ctivation has p ro tective effects on LPC-induced

d em yelina tion in cerebellar slices and th a t it m ight e xhibit its p ro tective effects via in h ib itio n o f pro-

V a lu e o f research

EBI2 is a fairly recently deorphanised receptor that has attracted much interest over the last 4-5

years. EBI2 is mainly expressed in the lymphoid tissue and the cells o f the immune system such as B

cells. For these reasons its function and expression in the immune system have been the main focus

of research. Astrocytes, among other functions, also serve immune functions in the CNS. They

present antigens to peripheral immune cells and release cytokines and chemokines that activate and

drive immune cell influx into the CNS. Even though astrocytes are necessary fo r a proper CNS

function and physiology, they have also been implicated in a range of neurodegenerative diseases

w ith an immune component such as m ultiple sclerosis, Alzheimer's disease or Parkinson's disease.

Moreover, cholesterol is a major component of myelin and the EBI2 agonists, oxysterols, are

cholesterol derivatives involved in its homeostasis. It is therefore warranted to investigate the

expression and function of EBI2 in the CNS and its involvement in astrocyte biology and myelination.

Therapies that control astrocyte activation may prove useful in managing neuroinflam matory or

neurodegenerative diseases and EBI2 might be a valuable tool via which inflam m atory signalling and

myelination can be controlled. This study explored the expression and function o f EBI2 in human and

mouse astrocytes and its role in myelination. The data showed th a t EBI2 is expressed in astrocytes

and th a t it plays im portant roles in the cell physiology. Activation o f EBI2 induced intracellular

signalling and cellular migration and dampened pro-inflam m atory signalling. Im portantly, this study

showed that astrocytes respond to immune challenge by regulating EBI2 and oxysterol expression.

The data also suggests that astrocytes communicate w ith the cells of the immune system, such as macrophages, via the EBI2 signalling system (the receptor and 7a25HC synthesising enzymes).

Im portantly, the data showed that EBI2 is involved in myelin development and can protect from LPC-

induced demyelination most likely via inhibition of pro-inflam m atory cytokine signalling. Taken

together, these studies support the possibility that EBI2 in astrocytes regulates inflammatory

responses and astrocyte migration. These results provide initial evidence fo r the use of EBI2 as a

novel drug target to altering astrocyte function in neuroinflam matory and neurodegenerative

Outputs

Papers

Rutkowska A., Sailer A.W. and Dev K.K. EBI2 regulates myelination state and protects from LPC-

induced dem yelination in organotypic cerebellar slice cultures. In preparation.

Preuss I., Rutkowska A., Dev K.K., and Sailer A.W. Signalling o f the oxysterol / EBI2 system in immune

cells. In preparation.

Rutkowska A., Sailer A.W. and Dev K.K. EBI2 regulates levels o f IL6 and TNFa in astrocytes and th e ir

signalling w ith macrophages. EMBO Reports, to be submitted.

Rutkowska A., Preuss I., Gessier P., Sailer A.W. and Dev K.K. EBI2 regulates intracellular signalling

and migration in human astrocytes. Glia, 10.1002/glia.22757.

Rutkowska, A., Elain, G., Jeanneau, K., Healy, L.M., M ir, A.K., Dev, K.K. SIP receptors regulate IL17-

induced levels o f IL6 in human astrocytes. Glia, in preparation.

Elain, G., Jeanneau, K., Rutkowska, A., Mir, A.K., Dev. K.K. The selective anti IL17A monoclonal

antibody Secukinumab (AIN457) attenuates IL17A-induced levels o f IL6 in human astrocytes.

Glia, 2014; 62(5):1098-1136.

Healy, L.M., Sheridan, G.K., Pritchard, A.J., Rutkowska, A., Mullershausen, F., Dev, K.K. Pathway

specific m odulation o f S lP l receptor signalling in rat and human astrocytes. British Journal o f Pharmacology, 2013; 169:1114-1129.

Doyle, P., Rohde, D., Rutkowska, A., Morgan, K., Cousins, G., McGee, H. Systematic review and m eta­

analysis of the impact of depression on subsequent smoking cessation in patients w ith

coronary heart disease: 1990-2013. Psychosomatic Medicine, 2014; 76:44-57.

Posters

Rutkowska, A., Preuss, I., Sailer, A.W., Dev, K.K. EBI2 regulates intracellular signalling and migration

in human astrocytes. 9th PENS Porum o f Neuroscience, Milan, Italy, July 5-9 2014.

Rutkowska, A., Sailer, A.W., Dev, K.K. Oxysterol-EBI2 signalling in astrocytes and th e ir role in

demyelination. Astrocytes in Health and Neurodegenerative disease, a jo in t Biochemical

Society/British Neuroscience Association Pocused Meeting, London, April 28-29 2014.

Rutkowska, A., Sailer, A.W., Dev, K.K. EBI2 directs astrocyte migration and protects from

demyelination. Poster presented at 3rd Annual Meeting Frontiers in Neurology Ireland,

Dublin, Ireland, 2013.

Preuss, I., Voshol H., Abraham Y., Lasbennes MC., Laurent S., Rutkowska A., Baumgarten B., Zhang J.,

Gessier P., Dev K.K., Seuwen K., Sailer A.W. Signalling o f the oxysterol/EBI2 system in immune

cells. Poster presented at 13th International Conference on Bioactive Lipids in Cancer,

Inflammation, and Related Diseases, Puerto Rico, 2013.

Elain, G., Jeanneau, K., Rutkowska, A., Dev, K.K., M ir, A.K. IL17A and TNFa regulate levels of

chemokines and cytokines in human astrocytes via MAP kinase and NFkB signalling pathways.

Poster presented at European Committee fo r Treatment and Research in M ultiple Sclerosis

(ECTRIMS), Copenhagen, Denmark, 2013.

Rutkowska, A., Dev, K.K., Sailer, A.W. Oxysterols stimulate astrocyte migration via the EBI2 receptor.

Poster presented at the 5th School o f Medicine Postgraduate Research Day, TCD, Dublin,

Rutkowska, A., Dev, K.K., Sailer, A.W. EBI2 receptor directs human astrocyte migration. Poster presented at the NSI Conference, RCSI, Dublin, 2012.

Rutkowska, A., Dev, K.K., Mosbacher, J., Deymier, C., & Muehlemann, A. The role of SIP receptors in LPS-induced cellular stress in microglia. Poster presented at the NSI Conference, Maynooth,

2011.

Awards

Best poster aw/ard fo r a poster presented at the 3rd Annual Meeting Frontiers in Neurology Ireland, Dublin, Ireland, 2013. Rutkowska, A., Sailer, A.W., Dev, K.K. EBI2 directs astrocyte migration and protects from demyelination.

Biochemical Society Student Travel Bursary (£300) to attend Astrocytes in Health and Neurodegenerative disease conference taking place in London on April 28-29 2014.

O verview

Epstein-Barr virus (EBV)-induced gene 2 (EBI2), also known as GPR1S3, is a G protein-coupled receptor (GPCR) which is highly expressed in peripheral blood mononuclear cells (PBMCs) (Birkenbach et a!., 1993, Benned-Jensen et al., 2012). EBI2 regulates B cell positioning w ith in the

lymphoid tissue and is crucial fo r launching appropriate T cell-dependent antibody response (Gatto

et al., 2009, Gatto et al., 2011, Kelly et al., 2011, Pereira et al., 2010). In addition to being crucial fo r launching correct humoral response, EBI2 is also involved in inflam m atory responses (Gatto et al., 2011, Gatto et al., 2009, Pereira et al., 2009). EBI2 is activated by oxysterols and signals through the pertussis toxin (PTx)-sensitive heterotrim eric G proteins of the Gi/o fam ily leading to a decrease in cyclic adenosine monophosphate (cAMP) production, calcium mobilization and stim ulation of the

extracellular-signal-regulated kinase (ERK) pathway (Hannedouche et al., 2011, Liu et al., 2011,

Benned-Jensen et al., 2013, Benned-Jensen et al., 2012).

Oxysterol 7a, 25-dihydroxycholesterol (7a25HC) and closely related compounds have been identified as high affinity agonists fo r EBI2 (Liu et al., 2011, Hannedouche et al., 2011). Oxysterols are oxygenated derivatives o f cholesterol and play im portant roles in metabolism o f vitamins and lipids, cholesterol trafficking, activation of nuclear hormone receptors, regulation o f inflam m atory

signalling pathways, and many more (Schroepfer, 2000, Leoni and Caccia, 2011, Russell, 2000).

Oxysterols have chemotactic properties inducing cell migration in vitro and in vivo (Liu et al., 2011, Hannedouche et al., 2011). Moreover, oxysterols have im m unom odulatory properties having both,

pro- and anti-inflam m atory effects depending on the oxysterol and cell type (Koarai et al., 2012, Aye

et al., 2012, Diczfalusy et al., 2009, Rosklint et al., 2002, Lemaire-Ewing et al., 2009, Moog et al., 1988).

W hile the role of EBI2 in the immune system is the main focus of research its expression and function in the cells of the central nervous system (CNS) have not been investigated. Astrocytes are one o f the four resident brain cells th a t serve a range o f physiological functions in the CNS. Among other roles, they are involved in CNS immune defence. They communicate w ith cells of the immune system via signalling molecules (for example by cytokines and chemokines) and cell-cell interactions

(for example by antigen presentation) (Kimelberg and Nedergaard, 2010, Ransom et a!., 2003).

Astrocytes also isolate inflamed or injured areas by form ing glial scars. They express a wide range of receptors and release various signalling molecules allowing them to be involved in both, normal and

pathophysiological processes in the CNS (Kimelberg and Nedergaard, 2010, Ransom et al., 2003).

Im portantly, these glial cells have been implicated in many neurodegenerative diseases such as m ultiple sclerosis (MS), Parkinson's disease (PD) and Alzheimer's disease (AD) (Lin et al., 1993, Knott et al., 2002, Sidoryk-Wegrzynowicz et al., 2010, Kimelberg, 2010, Sriram, 2011).

A quarter of the to ta l body cholesterol is located in the brain o f in oligodendrocytes, neurons and astrocytes (Leoni and Caccia, 2011). Cholesterol is the main building block o f myelin and is crucial for correct and tim ely myelin form ation (Nelissen et al., 2011). Astrocytes participate in myelination

during development by upregulating cholesterol synthesis (Pfrieger and Lingerer, 2011). Oxysterols

are also involved in cholesterol homeostasis and turnover and have been shown to play im portant

roles in myelination (Nelissen et al., 2011). In addition to being involved in normal myelin

development, oxysterols can have detrim ental effects on myelin and oligodendrocytes (Makoukji et

n eurod eg en erative diseases such as Alzheim er's disease or m ultiple sclerosis (Lutjohann e t a!., 20 0 0 , Leoni e t al., 2 0 0 2 ). U pregulated levels of oxysterols have been d etected in th e brains o f EAE animals and MS patients (D iestel e t al., 2 0 0 3 ). In spastic paraplegia gene 5 (SPG5) m utations in CYP7B1 gene lead to n europ ath y o f upper m o to r neurons as w ell as p eriventricular and subcortical w h ite m a tte r lesions (Schule and Schols, 20 1 1 , Biancheri e t a!., 2009).

1 EBI2 R e c e p to r

1.1 Structure and expression o f EBI2

EBV-induced gene 2 (EBI2, also known as GPR183) is a G protein-coupled receptor (GPCR) which belongs to th e largest and most studied fam ily of GPCRs called rhodopsin-like receptors (also known as fam ily A) (Birkenbach e t a!., 1993). EBI2 is located in chrom osom e 13 in region q 32.3 very close to GPR18 which is its closest hom ologue (Rosenkilde e t a!., 2 0 0 6 ). EBI2 also has some hom ology w ith GPR17, cysteinyl leukotriene receptors and purinergic receptors (e.g. PSY2, PSY6) (N o rreg aard e t a!., 2 0 1 1 ). Am ong the most prom inent characteristics o f EBI2 structure is th e lack o f a signal peptide. M o re interestingly, th e sequence encoding human EBI2 has tw o m ethionine codons in th e N-term inus, and it is currently unclear which one is responsible fo r initiation o f translation. The m urine sequences lack th e first m ethionine but th e y do not seem to have any functional consequences in term s of expression (R osenkilde e t a l., 20 0 6 , Birkenbach e t a!., 1993, N orregaard e t a l., 20 1 1 ). Figure 1.1 shows aligned hum an, rat and mouse sequences o f EBI2 and Figure 1.2 shows EBI2 phylogenetic tree.

EBI2 is expressed at th e cell surface m ainly in lymphoid tissue (spleen and lymph nodes) and peripheral blood m ononuclear cells (PBMC) mainly on B and T cells (Rosenkilde e t al., 2 0 0 6 ). EBI2 is upregulated during B cell m aturation and is expressed in recirculating B cells in th e spleen, lymph node and bone m arrow (Pereira e t a l., 20 0 9 ). EBI2 expression is highest in naive B cells and a fte r interaction w ith T helper cells, and it is alm ost undetectable in germ inal centre (GC) B cells (Rosenkilde e t al., 2006, N orregaard e t al., 2 0 1 1 ). EBI2 expression has also been found in granulocytes, dendritic cells, macrophages, monocytes, neutrophils, human platelets, tonsils and pancreas (Birkenbach e t al., 1993, Pereira e t al., 2 0 09, Am isten e t al., 2 0 08, Chan e t a l., 2010b, H annedouche e t al., 2011, Liu e t al., 2 0 1 1 , N orregaard e t al., 2 0 1 1 ). Figure 1.3 illustrates EBI2 expression in d ifferen t tissue and cells (adapted from Rosenkilde e t al., 2006).

1.2 Signalling o f EBI2

EBI2 is coupled exclusively to th e G| protein and w hen activated signals via tw o m itogen-activated protein (M AP) kinases, p38 and extracellular-signal-regulated kinase (ERK), as well as calcium and serum response e lem ent (SRE) transcription factor in a pertussis toxin (Ptx)-sensitive m anner. Notably, it does not signal via nuclear fa c to r o f activated T cells (NFAT) or nuclear factor kappa B (N FkB) (Liu e t al., 2 0 1 1 , Rosenkilde e t al., 2 0 0 6 , Benned-Jensen e t a l., 20 1 1 , Benned-Jensen e t al., 2 0 1 3 , H annedouche e t al., 2 0 11, D aug vilaite e t a!., 20 1 4 ). Figure 1.4 illustrates EBI2 signalling pathways. EBI2 is activated by several oxysterols and am ong th e m , 7 a , 25-dihydroxycholesterol (7a25H C ) has th e highest affinity and is th e most potent endogenous agonist fo r EBI2 w ith affinity ranging from 2 + /- 2 nM (H an ned o uch e e t a l., 2 0 11, Liu e t al., 20 1 1 ). O ther related oxysterols activate EBI2 w ith low er potency ranging fro m 5 + /- 4 nM fo r 7a27H C , 121 + /- 54 nM fo r 7P25HC, 3 1 0 + /- 261 nM fo r 7PHC, 1453 + /- 1163 nM fo r 7aHC, 3032 + /- 2645 nM fo r 25HC, to above 1 0,000 n M fo r cholesterol (H annedouche e t al., 2 0 1 1 ). In addition to agonists, th e re are currently tw o characterised EBI2 com petitive antagonists, NIBR189 and GSK682753A (Benned-Jensen e t a l., 20 1 3 , Gassier e t al., 20 1 4 ). Figure 1.5 shows th e chemical structure of EBI2 agonists and antagonists.

c h em o attractan t properties; indicating a ligand-induced receptor desensitization, most likely due to receptor internalization (Liu e t al., 20 1 1 , H annedouche e t aL, 2 0 1 1 ). In fact, 7a25H C induces EBI2 internalization a fte r as little as 10 m inutes o f tre a tm e n t w ith 7 a 2 5 H C (H annedouche e t a!., 2 0 1 1 , Liu e t al., 2 0 1 1 , Yi e t al., 2 0 1 2 ). O th e r related oxysterols such as 7a 2 7 H C , 7P25HC, 7aH C and 25HC also induce EBI2 expressing cell m igration although w ith much lo w er potency than 7a25H C (H an ned o uch e e t a l., 2011).

1.3 Oxysterols

Oxysterols are oxygenated derivatives o f cholesterol, which are involved in m etabolism o f vitamins and lipids, cholesterol homeostasis and trafficking, bile acid synthesis, apoptosis, necrosis, in flam m ation, im munosuppression, and many others (O taeg u i-A rrazo la e t a!., 2 0 1 0 , Bjorkhem and Diczfalusy, 2 0 02, S chroepfer, 2 0 0 0 , Leoni and Caccia, 2 0 11, Russell, 2 0 0 0 ). Oxysterols, o th e r than 7a25H C , are also agonists fo r nuclear horm one receptors such as liver x receptors (LXR) (Spann and Glass, 2 0 1 3 ). Agonism of LXR has effects on gene expression in brain cells such as astrocytes, for instance, by inducing synthesis o f ATP-binding cassette (ABC) transporters (W h itn e y e t al., 20 0 2 ). Oxysterols are fo rm ed via several pathways such as autoxidation, action of monooxygenase or by enzym atic or non-enzym atic lipid peroxidation (B jorkhem and Diczfalusy, 20 0 2 ). Oxysterols form ed by oxidation in th e ring have been found to have cytotoxic effects w hile oxysterols fo rm ed via enzym atic reaction ten d to have a range o f beneficial biological functions (O taegui-A rrazola e t al., 20 1 0 ). 7a25H C is synthesised from cholesterol through enzym atic action o f cholesteroi-25-hydroxylase (CH25H) follow ed by a cytochrom e P450 hydroxycholesterol 7-alpha-cholesteroi-25-hydroxylase (CYP7B1) and is degraded by c h olest-5-ene-3p,7a-d io l 3p-dehydrogenase (HSD3B7) (Yi e t al., 2012). Figure 1.5 shows th e synthetic pathways o f 7a25HC .

1.4 EBI2 function in the imm une system

EBI2 plays very im p o rta n t roles in th e im m une system by being involved in th e correct positioning o f

activated B cells in th e lym phoid organs and launching a pp ro priate a ntib od y response (Gatto et al., 2013, Kelly et al., 2011). In brief, chem okine re ce p to r typ e 5 (CXCR5) guides naive B cells to the follicles in th e lym phoid tissue. During norm al hum oral im m une response, s h o rtly a fte r B cell

rece pto r (BCR) engagem ent, activated B cells upregulate EB12 w hich tra n sie n tly guides th e m to the

o u te r regions o f the fo llicle, possibly to e n co un te r m ore antigens e ntering fro m circulation (Kelly et al., 2011). This effe ct is counteracted by strong signals fro m chem okine receptor typ e 7 (CCR7) (also know n as E B Il) w hich is upregulate a bo ut 6 to 10 hours a fte r BCR engagem ent. CCR7 directs B cells

to th e B zone - T zone (B-T) boundary w here th e y interact w ith cognate T helper cells (Kelly et al., 2011, Pereira et al., 2010, Pereira et al., 2009, Gatto et al., 2009, Gatto et al., 2011). CXCR5 and EB12 co ntin ue to signal c o n trib u tin g to equal d is trib u tio n o f B cells along th e B-T boundary ensuring

co rrect B cell to T cell interactio ns (Kelly et al., 2011). Subsequently, fo llo w in g CCR7 d ow nre gu la tio n and cluster o f d iffe re n tia tio n 40 (CD40) engagem ent, EBI2 fu n c tio n takes over and directs cells away

fro m th e B-T boundary and to w a rd s th e o u te r- and in te r- areas o f th e fo llicle (Kelly et al., 2011). From th ere, some cells m ove to th e extra fo llic u la r areas in CXCR4- and sphing osin e-l-p ho sp ha te

(S lP l)-d e p e n d e n t m anner, d iffe re n tia te into plasmablasts and m o u n t a rapid a ntib od y response.

Some cells how ever, m ove to th e fo llicle centre, d iffe re n tia te into GC B cells and undergo antibody

a ffin ity m atu ratio n, necessary fo r a lon g-te rm a ntib od y response (Kelly et al., 2011, Pereira et al., 2010, Pereira et al., 2009, Gatto et al., 2009, Gatto et al., 2011). The signals th a t are involved in d irectin g B cells to th e GC and stim u la tin g th em to leave th e fo llicle to m o u n t a s h o rt-te rm antibody

response are still unclear. Figure 1.6 illustrates th e sequence o f events described above.

EBI2 deficiency does not a ffe ct norm al GC response, fo llic u la r organization, lym phocyte num bers or

B cell m igration to th e B-T boundary th a t occurs w ith in hours o f BCR stim ulatio n (Pereira et al., 2009, Gatto et al., 2009). However, in th e subsequent stages o f th e hum oral response EBI2 d eficient B cells fa il to move to th e o u te r- and in te r-fo llic u la r regions and instead fa vo u r th e fo llic le centre

(Gatto et al., 2009, Pereira e t al., 2009). In add itio n, EBI2 deficiency results in a decreased early phase T cell d ependent a ntib od y defence show ing th a t EBI2 is crucial fo r a pp ro priate hum oral

response before th e involvem ent o f h ig h -a ffin ity GC plasma cells occurs (Gatto et al., 2009). On the o th e r hand, EBI2 overexpression o r suppression o f its d ow nre gu la tio n a fte r T cell in te ra c tio n results

in a dim inished GC response and increased B cell m igration to th e o u te r- and in te r-fo llic u la r regions

fo llo w e d by gre ate r plasmablast fo rm a tio n and increased conce ntra tion o f antibodies in th e early

phases o f T cell dependent a ntib od y response (Gatto et al., 2009, Pereira et al., 2009).

In CH25H (cholesterol-25-hydroxylase, a 7a25HC synthesising enzyme) d eficien t mice, activated B

cells fa il to m igrate to th e o u te r- and in te r-fo llic u la r regions and fa v o u r B-T boundary and central

areas o f th e fo llicle (Hannedouche et al., 2011). These CH25H-deficient m ice also show a th re e-fo ld decrease in th e e xten t o f th e ir a ntib od y response. On th e o th e r hand, saturating the

m icro e n viro n m e n t w ith 7a25HC o r in h ib itio n o f CYP7B1 (hydroxycholesterol 7-alpha-hydroxylase,

th e second 7a25HC synthesising enzyme) results in equal d is trib u tio n o f B cells in th e lym phoid

tissue suggesting th a t 7a25HC g ra die nt is crucial fo r app ro priate localization o f B cells in the

lym phoid tissue (Hannedouche et al., 2011, Liu et al., 2011). Thus, to ensure co rrect fu n c tio n in g o f th e hum oral a ntib od y response th e g ra die nt o f 7a25HC has to be tig h tly co ntrolle d. Since B cells do

n o t them selves express the 7a25HC synthesising (CH25H and CYP7B1) nor th e degrading enzyme

lym phoid strom al upregulate th e levels of 7 a25H C via CH25H, CYP7B1 enzymes and follicular

dendritic cells dow nregulate th e levels via HSD3B7 enzym e (Yi e t al., 2012).

EBI2 is involved in various stages o f th e hum oral response exerting a w ide range of effects (Kelly e t

al., 2 0 1 1 ). As G atto and colleagues (2009) suggested, regulation of EBI2 expression may be a

p ow erfu l tool to control B cell localization w ithin secondary lymphoid tissue. This m ay be achieved

via th e B cell lym phom a 6 (BCL-6) transcriptional repressor and th e non-canonical N F-kB pathw ay

which have been found to regulate EBI2 expression (G a tto e t al., 2 0 0 9 ). It has been shown th a t

BCR-triggered NF-kB activation transiently induces upregulation o f EBI2 in naive B cells and th a t BCL-6

triggers dow nregulation of EBI2 in GC B cells (S haffer e t a l., 2 0 0 0 , G lynne e t a l., 2 0 0 0 , G atto e t al.,

2 0 0 9 ). EBI2 expression has also been found to be regulated by cytokines such as interleukin 4 (IL4),

IL6 and ILIO (S haffer e t al., 20 0 0 , Lam e t al., 2 0 0 8 , G a tto e t al., 20 0 9 ).

EBI2 expression and function have so fa r only been investigated w ith regards to th e cells o f the

im m une system and th e receptor's expression and function in th e central nervous system remains

currently unknown. H ow ever, studies suggest th a t dysregulated expression o f EBI2 or o f its related

oxysterols have been im plicated in a n um ber o f CNS diseases such as Alzheim er's disease or m ultiple

sclerosis (Papassotiropoulos e t al., 20 0 0 , B jorkhem , 20 0 6 , Fuku m o to e t al., 2 0 0 2 , Koldam ova e t al.,

2 0 0 3 , Lutjohann e t a l., 2 0 0 0 , V aya and Schipper, 2 0 0 7 , Vega e t a l., 20 0 3 ). It is th e re fo re w arranted

to investigate th e role o f EBI2 in th e CNS as w ell as th e im m une system. EBI2 involvem ent in disease

Human MDIOMANNFTPPSATPOGNDCDLYAHHSTARIVMPLHYSLVFIIGLVGNLLALVVIVONRKKIN 64

Mouse

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Rat

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STTLYSTNLVISDILFTTALPTRIAYYAMGFDWRIGDALCRITALVFYINTYAGVNFMTC 124

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LSIDRFIAVVHPLRYNKIKRIEHAKGVCIFVWILVFAOTLPLLINPMSKOEAERITCMEY 184

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Figure 1.1 H um an, m ouse, and ra t EBI2 sequences. Alignm ent of hum an, mouse, and rat EBI2

sequences (h ttp ://b la s t.n c b i.n lm .n ih .g o v /B la s tA lig n .c g i). Amino acid residues th a t are identical in

hum an EBI2 are indicated by dashes; differences are shown. The transm em brane dom ains are

Monkey (0.0003)

Human (0.0024)

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Chicken (0.1644)

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other EBI2 activating oxysterols. (c) Chemical structure of EBI2 antagonists, NIBR189 and

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2 Astrocytes

2.1 Astrocyte origins and types

Astrocytes are one o f the three well known types of resident CNS glial cells alongside microglia and

oligodendrocytes (and not withstanding others such as glial antigen 2 (NG2) positive glia). Astrocytes

are homeostatic cells of the CNS characterised by a star-shaped morphology and spreading

numerous extended processes that surround neurons and blood vessels (Wang and Bordey, 2008,

KImelberg, 2010, Butt et a!., 2002). They are non-excitable and are identified by interm ediate

filaments called glial fibrillary acidic proteins (GFAP) (Wang and Bordey, 2008, KImelberg, 2010). Of

importance, it has to be noted that not all astrocytes have a star-shaped morphology or express

GFAP (Kimelberg, 2004).

Astrocytes originate from neural progentitors that give rise to both, macroglia (astrocytes and

oligodendrocytes) and neurons (Verkhratsky and Butt, 2013). The neural progenitors differentiate

into radial glial cells in the ventricular zone and can give rise to astrocytes, neurons and some

oligodendrocytes. Most oligodendrocytes, however, are generated in distinct areas of the CNS from

specific glial precursors. Astrocytes, on the other hand, originate from both, radial glial cells and glial

precursors (Verkhratsky and Butt, 2013).

Astrocytes are an exceptionally varied cell type and many subtypes that distinguish one type from the other can be named based on th e ir function, cellular presence, morphology, gene expression,

physiological properties and response to stim uli (Verkhratsky and Butt, 2013). One classification

divides these cells into type 1 and type 2 astrocytes. Type 1 astrocytes are defined as fibroblast-type cells that originate early in development and secrete growth factors that, in turn, can stim ulate the

growth o f type 2 astrocytes (Raff et al., 1983, Armstrong et al., 1990). Type 2 astrocytes can

differentiate into 0-2A progenitor cells and give rise to oligodendrocytes (Raff et al., 1983,

Armstrong et a!., 1990). Other classification divides astrocytes into protoplasmic astrocytes, found in

the grey m atter of the brain and the spinal cord, and fibrous astrocytes, found in the w hite m atter in

the brain, the spinal cord, optic nerve and retina (Verkhratsky and Butt, 2013). These are the most

numerous subtypes o f astrocytes, however, there are other types which include radial glia and other

radial glia-type cells such as M uller glia in the retina and Bergmann glia in the cerebellum

(Verkhratsky and Butt, 2013). In addition, there are other, much less abundant subtypes which

include: perivascular and marginal astrocytes, velate astrocytes o f the cerebellum and olfactory bulb,

interlam inar and polarised astrocytes found in the primate cortex, and varicose projection astrocytes

present only in human brains (Verkhratsky and Butt, 2013). Other subtypes include tanacytes found

in the periventricular organs, hypothalamus and the spinal cord, pituicytes in the pituitary gland, and

retinal and ventrical cells such as ependymocytes, choroid plexus cells and retinal pigment epithelial

cells (Verkhratsky and Butt, 2013).

2.2 Astrocyte functions

Astrocytes are a very abundant cell type in the brain and serve a range o f physiological functions. For

instance, they control neuronal m aturation, form and maintain synapses, uptake neurotransmitters,

release antioxidants and are responsible fo r extracellular ion buffering (Kimelberg and Nedergaard,

2010, Wang and Bordey, 2008, Ransom et al., 2003, Kimelberg, 2010). Astrocytes prom ote

myelination and speed up myelin wrapping around axons and form ation o f compact myelin during

expression is necessary fo r correct nnyelination and blood-brain barrier form ation (Liedtke e t al., 19 9 6 ). In addition to supporting neurons, astrocytes also play a role in m aintenance of th e BBB and com m unication w ith th e im m u ne system via antigen presentation and cytokine/chem okine production (K im elberg and N e d erg aard , 2 0 1 0 , W an g and Bordey, 2 0 0 8 , Ransom e t al., 2003, K im elberg, 20 1 0 ). They also regulate synaptic transmission by processing and integrating neuronal activity and coordinating calcium waves, suggesting astrocyte involvem ent in intercellular com m unication (K im elberg and N edergaard , 2 0 1 0 , W an g and Bordey, 2 0 0 8 , Ransom e t al., 2003, K im elberg, 20 1 0 ). Table 1.1 lists various types o f astrocyte functions.

Astrocytes also express a w id e range o f receptors such as G protein coupled receptors (GPCRs), ionotropic receptors as well as receptors fo r grow th factors, chemokines, cytokines, steroids, toll-like receptors (TLRs), and, them selves, release neurotransm itters, grow th factors and o th e r signalling m olecules allowing th em to be involved in both normal and pathophysiological processes taking place in th e CNS (K im elberg and N edergaard , 2 0 1 0 , W an g and Bordey, 2 0 0 8 , Ransom e t al., 2003, K im elberg, 20 1 0 ). Figure 1.7 illustrates some o f th e large rep ertoire o f proteins expressed by astrocytes.

During CNS dam age or injury astrocytes facilitate healing of th e injured tissue, form glial scars to lim it th e exten t o f dam age and release pro-inflam m atory cytokines and chemokines leading to activation of microglia and influx o f leukocytes into th e CNS. Astrocytes have also been found to present myelin antigens to T cells in MS (Fontana e t al., 1984). Although others have shown th a t only microglia, and not astrocytes, present myelin basic protein (M BP) antigens to T cells and astrocytes are involved in suppression of T cell proliferation and release of an ti-inflam m atory cytokines but not in phagocytosis (M a ts u m o to e t al., 1992).

2.3 S im ilarities and differences b e tw e e n ro d e n t and hum an astrocytes

Part of o ur knowledge about astrocyte biology and function comes from in vitro studies th a t use mouse or rat prim ary cells. W h ile hum an and rodent brain cells share many similarities th e re are also differences b etw een species th a t need to be taken into account w hen conducting research (O b e rh e im e t al., 20 0 9 ). For instance, both rodent and human astrocytes respond to m etabotropic agonists by intracellular Ca^* changes. On th e o th e r hand, hum an astrocytes are m ore varied in m orphology and are approxim ately 2.6-fold larger than rodent astrocytes (O berheim e t al., 2009). M u rin e and hum an astrocytes also express d ifferen t receptors. For instance, mouse astrocytes express a w ide range o f to ll-like receptors (TLRs) while human astrocytes preferentially express TLRS and do not express TLR4 receptors (Farina e t al., 2 0 0 5 ). These differences in cell biology may account fo r th e fact th a t m any compounds fail w hen tested in humans in spite o f excellent th e ra p e u tic effects in animals. Table 1.2 lists th e similarities and differences b etw een rodent and hum an astrocytes (O b e rh e im e t al., 20 0 9 ).

2 .4 Astrocyte in v o lv e m e n t in disease

inflam m atory infiltrates (D'Amelio et al., 1990, Morcos et al., 2003, Smith and Sommer, 1992). Figure 1.8 illustrates how astrocytes exacerbate the pathophysiology of m ultiple sclerosis. Astrocytes also contribute to neurodegeneration in Parkinson's disease via release of toxic molecules such as l-M ethyl-4-phenylpyridinium (MPP^) that is taken up by dopaminergic neurons and causes neurotoxicity (Rappold and Tieu, 2010). In Alzheimer's disease, (3-amyloid plaques activate astrocytes, which in turn secrete pro-inflam m atory cytokines that exacerbate the neuronal injury taking place during Alzheimer's disease (Johnstone et a!., 1999).

There is a bi-directional communication between astrocytes and peripheral immune cells via cytokine/chemokine system and other signalling molecules. At the site of injury, astrocytes release cytokines and chemoklnes to activate resident immune cells and induce influx o f leukocytes from the periphery (Miljkovic et al., 2011, Miljkovic et al., 2007). They stimulate interferon gamma (IFN-y) and IL17 production by T helper 1 (T h l) and T h l7 cells in m ultiple sclerosis plaques and respond to the stim ulation w ith these cytokines by increasing expression of inducible nitric oxide (iNOS), NO and other pro-inflam m atory mediators such as IL6 implicated in m ultiple sclerosis pathogenesis (Miljkovic et al., 2011, Bo et al., 1994, Miljkovic et al., 2007). Increased levels of IL6 enhance its own production in a positive feedback loop and also drive IL17 synthesis and release (Ma et al., 2010, Meares et al., 2012, McGeachy et al., 2007). Some studies have suggested that astrocytes also drive the development o f EAE by phagocytosing myelin and presenting myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG) and proteolipid protein (PLP) antigens to CD4* T cells. However, the role o f astrocytes and th e ir phagocytosing capabilities remain a topic of debate in the field (Tan et al., 1998, Kort et al., 2006, Lee et al., 1990, Fontana et al., 1984).