Analysis of the impact of SIGIRR on glial function

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LEABHARLANN CHOLAISTE NA TRIONOIDE, BAILE ATHA CLIATH TRINITY COLLEGE LIBRARY DUBLIN OUscoil Atha Cliath The University of Dublin

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Analysis of the impact of SIGIRR on glial function

Donal Carney

A thesis submitted to Trinity College Dublin for the degree o f Doctor

o f Philosophy

Supervisor: Professor Marina A. Lynch

Department o f Physiology

Trinity College Dublin

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TRINITY C O L L E G E ^ 2 A MAY 2013

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I.

Declaration

T his th esis is su bm itted by the undersigned for the degree o f D octor o f P hilosophy at the U niversity o f D ublin. I declare that this w o rk is entirely m y own. T his w ork has not been subm itted, in w hole or in part, to this o r any o ther university for any o th er degree. The au th o r gives perm ission to the library to lend o r copy this w o rk upon request.

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II.

Abstract

M a n y n eu ro d e g e n e r a tiv e d is e a s e s are a s s o c ia te d w ith a sh ift to w a r d s a p ro -in fla m m a to r y e n v ir o n m e n t w ith in th e C N S , and th is is b e lie v e d to co n trib u te to th e p r o g r e ssiv e d e c lin e in c o g n it iv e fu n c tio n a sso c ia te d w ith th e s e d is e a s e s . A c tiv a te d g lia l c e lls are th o u g h t to be th e d riv in g fo rc e b eh in d th e d e v e lo p m e n t o f a p r o -in fla m m a to r y e n v ir o n m e n t, and e n d o g e n o u s m o d u la to r s o f g lia l a c tiv ity h a v e e m e r g e d as targets fo r th e treatm en t o f c h ro n ic n e u r o in fla m m a tio n . T h e o b je c tiv e o f th is th e s is w a s to a n a ly se th e a b ility o f o n e su ch m o le c u le , S IG IR R , to act as a m o d u la to r o f g lia l a ctiv a tio n .

T h e e f f e c t s o f L P S , I L - lp , P ani3C S K4 and A(3 o n m arkers o f m ic r o g lia l a c tiv a tio n and p ro d u c tio n o f c y to k in e s w a s a s s e s s e d in m ix e d g lia and iso la te d a str o c y te s an d m ic r o g lia p repared fro m w ild ty p e and S lG I R R -d e f ic ie n t m ic e . N e x t , the e f f e c t o f an a cu te in tra h ip p o ca m p a l in je ctio n o f A p in y o u n g and m id d le -a g e d , w ild ty p e and S IG IR R - d e fic ie n t m ic e w a s a s s e s s e d . F in a lly , 1 L -1 F 5 , S IG IR R /F c and T R E M - l/F c w e r e a s s e s s e d for th eir a b ility to p ro m o te S IG IR R e x p r e s s io n a n d /o r a ctiv a te th e recep tor.

M ix e d g lia and a str o c y te s from S lG lR R - d e f ic ie n t m ic e e x h ib ite d in crea sed b asal e x p r e s s io n o f T L R 2 and T L R 4 co m p a r e d to c e lls from w ild ty p e m ice . It is p ro p o sed that th is is r e s p o n s ib le for th e o b se r v e d e x a g g e r a te d p r o d u c tio n o f lL -6 and T N F - a in m ix e d g lia fro m S lG lR R -d e f ic ie n t m ic e co m p a r e d w ith w ild ty p e m ic e in r e sp o n se to L P S , I L - i p , P a m3C S K4 and A p . Iso la ted m ic r o g lia , but n ot a str o c y te s, from S lG lR R -d e f ic ie n t m ic e , e x h ib ite d an d an e x a g g e r a te d p ro d u c tio n o f IL -6 c o m p a re d w ith w ild ty p e m ic e in r e sp o n se to L P S , h o w e v e r th is w a s s e e n in b o th a s tr o c y te s and m ic r o g lia in r e sp o n se to P a m3C S K4. P re-treatm en t o f m ix e d g lia w ith an a n ti-T L R 2 a n tib o d y atten u ated th e A P -in d u c e d in cr ea se in IL -6 and T N F - a s u g g e s t in g that T L R 2 a c ts as a re ce p to r for A p.

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dose-dependent manner. Pre-treatment with T R E M -l/F c attenuated the LPS-induced increase in T N F -a from mixed gHa and isolated m icroglia in a dose-dependent manner. T R E M -l/F c reversed the LPS-induced decrease in SIGIRR m RNA expression.

IL -lp induced the mRNA expression o f markers o f microglial activation, C D l l b and CD68, in mixed glia, which was exaggerated in cells prepared from SIGlRR-deficient

mice. Similarly the IL-ip-induced increases in lL-6, T N F -a and MPC-1 in mixed glia were enhanced in cells prepared from SIGlRR-deficient mice. Pre-treatm ent with IL-1F5 was found to attenuate the IL -lp-induced increase in IL-6 in mixed glia prepared from wildtype mice but this effect was absent in cells prepared from SIG lRR -deficient mice.

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III.

Acknowledgements

I would like to express my sincere gratitude to my supervisor, Prof. M arina Lynch, for her help and guidance throughout my PhD, and for being an am azing teacher since 1 first attended one o f her lectures six years ago.

I ’d also like to thank all the M AL lab post-docs, past and present, w ho have been so generous w ith their time and willing to help w hen 1 needed it. I would also like to thank all the staff from the department o f Physiology for all their help.

1 would particularly like to thank Dr. A nthony Lyons for introducing me to the lab and for keeping an eye out for me ever since.

I ’d like to thank my office buddies for all the cups o f tea and the chats; Jim m y for teaching me about Cham pionship, and Rashers, for sharing my Game o f Thrones obsession. I have to especially thank Steph and Tara w h o ’ve been with me through the whole thing, and, o f course, my fellow Physiology graduates, Lolly and Suzie. We made it!

I would like to thank Gillian Muirhead for all her help with my molecular work, and my students, Ellie and Jeff, for their hard w ork and for being constant sources o f entertainment!

I ’d like to give a big thank you to Luke and Ronan for all the craic over the last three years.

I’d like to thank my parents and my brothers for the never-ending encouragem ent and support t h e y ’ve show n me.

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IV.

Abbreviations

AD A lzh e im e r’s disease

A PP A m yloid p recu rso r protein

AP

A m yloid-beta peptide

A N O V A A nalysis o f variance

A PC A ntigen presenting cells

A poE A polipoprotein E

B A C E P-A PP C leaving Enzym e

BBB B lood brain barrier

BSA B ovine serum album in

C N S C entral nervous system

C SF C erebrospinal fluid

CD C luster o f differentiation

cD N A C om plem entary deoxyribonucleic acid

CIH2O D eionised w a te r

Da D alton

DC D endritic C ell

DNA D eoxyribonucleic acid

dN T P D eoxyribonucleotide triphosphate

D M EM D ulb ecco ’s m odified eagle m edium

EL ISA Enzym e linked im m unosorbent assay

EA E E xperim ental autoim m une encephalom

Epo E rythropoietin

FBS Foetal bovine serum

Fc F ragm ent, crystalisable

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G F P G r e e n flu o re sc e n t pro tein

G M - C S F G r a n u l o c y t e - m a c r o p h a g e c o lo n y - s tim u la tin g facto r ( G M - C S F ) H B S S H a n k ’s b a la n c e d salt solution

H IV H u m a n i m m u n o d e f ic ie n c y virus H M G B I H ig h m o tility g r o u p b o x 1 H R P H o r s e radish p e ro x id a s e

IC A M In tra c e llu lar a d h e s io n m o le c u le

IkK IkB kinase

iK B a N u c l e a r fa c to r o f k a p p a light p o ly p e p tid e g e n e e n h a n c e r in B -cells inh ib ito r, alpha

I L - l R A c P 1L -1 R a c c e s s o r y pro tein IR A K IL -1 R a ss o c ia te d k in a se s Ig Im m u n o g lo b u lin

IFN -y In te r f e r o n - g a m m a

IL Interleu k in

I L - I R lnterleukin-1 re c e p to r

1L-1F5 Interleukin-1 fa m ily m e m b e r 5 lC A M -1 In tra c e llu lar a d h e n s io n m o l e c u l e - 1 I R A K Interleukin-1 re c e p to r a sso ciated kinase i.h. I n tra h ip p o c a m p a l

i.p. In tra p e rito n e al i.v. I n tra v e n o u s ly

J N K Ju n N - te r m in a l kinase J A K J a n u s K inase

k Kilo

-/- K n o c k o u t

L L igand

L PS L ip o p o ly s a c c h a r id e L T P L o n g - te r m p o te n tia tio n

M M o la r

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MAPK M itogen-activated protein kinase

M -CSF Macrophage colony-stim ulating factor

MHC class 11 Major histocom patibility com plex class II

m RN A M essenger ribonucleic acid

M yD 88 M yeloid differentiation primary response gene 88

1^ Micro

ml M illiliter

mm M illimetre

MAPK M itogen-activated protein kinases

mM Molar

MS M ultiple sclerosis

M yD 88 M yeloid differentiation factor 88

n Number

N F -kB Nuclear factor kappa-light-chain-enhancer o f activated B cells

Nm Nanometre

NK Natural killer

NO Nitric oxide

NG S Normal goat serum

N H S Normal horse serum

N F-kB Nuclear factor-KB

Pam3Csk4 Pam3CysSerLys4

PAM Ps Pathogen associated moleculare patterns

PRRs Pathogen recognition receptors

PBS Phosphate buffered saline

PBS-T Phosphate buffered saline containing tween

PCR Polymerase chain reaction

PD Parkinson’s D isease

RNase Ribonuclease

RNA Ribonucleic acid

ROS Reactive oxygen species

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S I G I R R S in g le - im m u n o g lo b u lin interleukin-1 r e c e p to r related S E M S ta n d a rd erro r o f m ean

S tr e p - H R P S tre p ta v id in -h o rs e ra d is h p e ro x id a s e linked H2SO4 S u lp h u ric acid

T A B l T G P - b e ta a c tiv ated k in ase 1 T I R D o m a in T o ll/in te r le u k in - 1 r e c e p to r d o m a in T M B T e tr a m e th y lb e n z id in e

T N F T u m o u r n ecro sis fa c to r T N F R T N P r e c e p to r

T R A P T N P r e c e p to r a sso c ia te d facto r

T R E M - 1 T rig g e rin g re c e p to r e x p re s s e d o n m y e lo id c e lls 1 T R I P T l R - d o m a i n - c o n t a i n i n g a d a p te r - in d u c in g in terferon-p T L R T oll like r e c e p to r

T g T ra n s g e n ic

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V.

3 .3 .5 In v e stig a tin g th e e f f e c ts o f I L - i p o n a str o c y te s prepared from w ild ty p e and S I G I R R " m i c e ...5 7 3 .3 .6 In v e stig a tin g th e e f f e c t s o f I L - i p o n m ic r o g lia prepared fi-om w ild ty p e and S IG IR R " m i c e ...5 7 3 .4 . D is c u s s i o n ... 71

3 .4 .1 . O v e r v i e w ... 71

3 .4 .2 . T h e e f f e c t o f L P S treatm en t o n m ix e d g lia from w ild ty p e and SIGIRR'^' m i c e ... 71

3 .4 .3 . T h e e f f e c t o f L P S treatm en t on a str o c y te s from w ild ty p e and S IG IR R ' " m ic e ... 72

3 .4 .4 . T h e e f f e c t o f L P S treatm en t o n m ic r o g lia from w ild ty p e and S I G I R R " m i c e ... 73

3 .4 .5 . T h e e f f e c t o f I L - I p treatm en t o n m ix e d g lia from w ild ty p e and SIG IR R "' m i c e 7 4 3 .4 .6 . T h e e f f e c t o f I L - i p treatm en t o n astroc>1es from w ild ty p e and S IG IR R " m i c e ... 75

3 .4 .7 . T h e e f f e c t o f I L - i p treatm en t o n m ic r o g lia from w ild ty p e and SIG IR R "' m i c e ...75

C h ap ter 4: A n a ly s is o f th e e f f e c t s o f Pam sC SIC t and A p on g lia l c e lls fi-om w ild ty p e and S IG IR R -d e fic ie n t m ic e ...7 7 4.1 I n tr o d u c t io n ... 7 8 4 .2 . M e t h o d s ...7 9 4 .3 . R e s u l t s ... 81

4 .3 .1 . In v e stig a tin g th e e f f e c t o f P a m3C S K4 o n g lia l c e lls prepared from w ild ty p e and SIGIRR"''* m ic e ...81

4 .3 .2 . In v e stig a tin g th e e f f e c t o f P a m3C S I< 4 on iso la te d a str o c y te s and m ic r o g lia p rep ared fro m w ild ty p e and SIG IRR " ' m ic e ...81

4 .3 .3 . In v e stig a tin g th e e f f e c t o f A p o n g lia l c e lls prepared from w ild ty p e and S IG IR R '^ 'm ic e ... 82

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4 .4 .1 . O verview 94

4 .4 .2 . The effect o f Pam3C SK4 treatm ent on m ixed glia prepared from w ild ty p e and SIG IR R " m ic e ...9 4

4 .4 .3 . The effe ct o f Pam3C SK4 treatm ent on astrocytes and m icroglia prepared from

w ild typ e and SIGIRR'^' m ic e ... 95

4 .4 .4 . The effect o f A p treatm ent on m ixed g lia prepared from w ild typ e and S IG IR R '' m ic e ... 97

Chapter 5: A n alysis o f the e ffect o f intrahippocam pal injection o f A p in you n g and m iddle-aged, w ildtype and S IG IR R -deficient m i c e ... 9 9 5.1. In tro d u ctio n ...100

5.2. M e th o d s... 101

5.3. R e s u lts ... 103

5.3.1. The e ffect o f A P and anti-T L R 2 antibody on m ixed g lia l c e l l s ... 103

5.3.. The effect o f age, A p and ab sen ce o f SIG IR R on cytok in es in cortical and hippocam pal tissu e ...103

5 .3 .3 . The effec t o f age, A p and ab sen ce o f SIG IR R on receptors in cortical and hippocam pal t i s s u e ... 103

5 .3 .4 . The effe ct o f age, A p and ab sen ce o f SIG IR R on markers o f glial activation in cortical and hippocam pal t i s s u e ...104

5 .3 .5 . The e ffe c t o f age, A p and ab sen ce o f SIG IR R on N F -kB activation in cortical and hippocam pal t is s u e ...104

5.4. D isc u ssio n ...113

5 .4 .1 . O v e r v ie w ...113

5 .4 .2 . The effec t o f b lock in g T L R -2 on A P -induced cytok in e production in m ixed g lia .... 113

5 .4 .3 . The effe c t o f aging on glial fu n c tio n ...114

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5.4.5. The effect o f age, A p injection and SIGIRR deficiency on cytokine production

in cortical and hippocampal tissue...115

5.4.6. The effect o f age, A(3 injection and SIGIRR deficiency on pro-inflam m atory receptor expression in cortical and hippocampal tissue...116

5.4.7. The effect o f age, A p injection and SIGIRR deficiency on markers o f m icroglial activation in cortical and hippocampal tissu e ... 117

5.4.8. The effect o f age, A p injection and SIGIRR deficiency on N F -kB signaling in cortical and hippocampal tissu e ... 118

Chapter 6: Analysis o f the effects o f IL-1F5, SIGIRR/Fc and T R E M -l/F c on stimulated glial cells... 120

6.1. Introduction... 121

6.2. Methods...123

6.3. Results... 125

6.3.1. Investigating the effect o f 1L-1F5 on IL-1 P-treated glial cells prepared from wildtype and S IG IR R " mice...125

6.3.2. Investigating the effect o f SIGIRR/Fc on LPS and IL-ip-treated glial cells... 126

6.3.3. Investigating the effect o f T R E M -l/F c on LPS-treated glial c e lls ...127

6.3.4. Molecular cloning o f human SIGIRR gene into mammalian vector... 128

6.4. Discussion... 144

6.4.1. O ve rv ie w ...144

6.4.2. The effect o f IL-1 F5 on IL-ip -in du ce d cytokine production from mixed g lia ... 144

6.4.3. The effect o f SIGIRR/Fc on LPS and IL-1 P-induced cytokine production from mixed g lia ... 146

6.4.4. The effect o f T R E M -l/F c on LPS-induced cytokine production from mixed glia ... 147

6.4.5. Transfection o f BV2 cells w ith vector containing s ig ir r gene... 148

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7.1. O v e rv ie w ...152

7.2. SIGIRR deficiency and glial cell fu n ctio n ... 152

7.3. The role o f SIGIRR as a m odulator o f inflammation in v iv o... 155

7.4. SIGIRR as a target for anti-inflam m atory th era p eu tics... 157

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VI.

Table of Figures

Figure 1.1. The Interleukin-1 Receptor/Toll-Like Receptor S u p e rf a m ily ... 13

Figure 1.2. IL-1R1/TLR4 signaling p a t h w a y ... 14

Figure 1.3. Formation o f am yloid-P p ep tid es...20

Figure 1.4. SIGIRR negatively regulates 1L-1R1/TLR4 signaling p a t h w a y ... 28

Figure 1.5. Chronic neuroinflammation in aging and neurodegenerative d isease...31

Figure 2.3. Summ ary o f molecular cloning p r o to c o l...49

Figure 3.1. Results s u m m a r y ...59

Figure 3.2. TLR4 and S lG lR R expression in mixed glia were modulated by LPS and S lG lR R defic ien cy ...60

Figure 3.3. LPS induced cytokine production in mixed glia w as exaggerated in cells prepared from SIGIRR' m i c e ...61

Figure 3.4. TLR4 expression was increased in astrocytes prepared from S IG IRR " m ice... 62

Figure 3.5. LPS induced cytokine m R N A expression in astrocytes was exaggerated in cells prepared from SIG IR R ' " m ice... 63

Figure 3.6 LPS-induced cytokine production in microglia was exaggerated in cells prepared from SIGIRR'^' m ice...64

Figure 3.7. I L - l R l and S IG IR R expression in mixed glia were modulated by I L - i p ...65

Figure 3.8. Expression o f markers o f microglial activation was increased in mixed glia prepared from S I G I R R '‘ m ice...66

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Figure 3.10. IL-l|3-induced cytokine m R N A expression in astrocytes was exaggerated in

cells prepared from S IG IR R " mice...68

Figure 3.11. I L - ip had no effect on microglia prepared from wildtype or S IG IR R " m ic e 69 Figure 3.12. U n ifyin g m echanism ... 70

Figure 4.1. Results sum m ary... 84

Figure 4.2. TLR 2 expression was increased in mixed glia prepared from S IG IR R " mice...85

Figure 4.3. PamsCSlC* induced CD40 expression in mixed g lia ... 86

Figure 4.4. Pam3CSI<4- induced cytokine production in mixed glia was exaggerated in cells prepared from S IG IR R " m ice ... 87

Figure 4.5. TLR2 expression is increased in astrocytes prepared from SIGIRR "m ice...88

Figure 4.6. PamsCSIQ induced cytokine release from astrocytes was exaggerated in cells prepared from S IG IR R " m ic e ... 89

Figure 4.7. FamsCSlQ-induced release o f IL-6 from microglia was exaggerated in cells prepared from S IG IR R '' m ic e ... 90

Figure 4.8. A p induced CD40 expression in mixed glia...91

Figure 4.9. Ap-induced cytokine production in mixed glia was exaggerated in cells prepared from SIGIRR " m ic e ... 92

Figure 4.10. U nifying m echanism ... 93

Figure 5.1. Blockade o f TLR 2 attenuated the AP-induced increase in cytokine release from mixed g lia ... 106

Figure 5.2. There was no effect o f age, A p or SIGIRR deficiency on m RN A expression o f lL -6 in cortical and hippocampal tissue from wildtype and SIG IR R ' mice... 107

Figure 5.3. m R N A expression o f receptors for AP was increased in cortical and hippocampal tissue prepared from SIGIRR "m ice... 108

Figure 5.4. There was no effect o f age, A p or SIGIRR deficiency on m RN A expression o f CD40 in cortical and hippocampal tissue from wildtype and SIGlRR''^"mice... 109

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Figure 5.6. Phosphorylation o f iK B a w as increased in hippocampal tissue from S IG IR R ’ ’ m i c e ...1 1 1

Figure 5.7. Unifying m e c h a n i s m ... 112

Figure 6.1. Results s u m m a r y ... 130

Figure 6.2. IL-1F5 attenuated IL-6 release in mixed glia prepared from wildtype mice but not SIG IRR " m i c e ... 131

Figure 6.3. IL-1F5 had no effect on m R N A expression o f I L - l R l o r S lG lR R in mixed glia from wildtype and S I G I R R " m ic e ...132

Figure 6.4. S lG IR R/F c attenuated the LPS-induced increase in m RNA expression o f C D l lb from mixed g lia ...133

Figure 6.5. S lG lR R /F c attenuates the LPS-induced increase in cytokine release from mixed g l i a ...134

Figure 6.6. SIG IRR/Fc had no effect on the m R N A expression o f SIGIRR in mixed glia...135

Figure 6.7. SIGIRR/Fc failed to attenuate the lL-l|3-induced increase in m RN A expression o f CD 40 from mixed glia... 136

Figure 6.8. SIGIRR/'Fc attenuated the IL-l(3-induced increase in IL-6 from mixed g l i a ...137

Figure 6.9. T R E M -I/F c attenuated the LPS-induced increase in T N F - a from mixed g l i a 138 Figure 6.10. T R E M - l/F c reversed the LPS-induced decrease in m R N A expression o f SIGIRR in mixed glia...139

Figure 6 . 1 1. T R E M - l/F c attenuates the LPS-induced increase in release o f T N F -a from m icroglia... 140

Figure 6.12. Original S IG IRR Insert from C om mercial Cloning V ector p E N T R 2 1 1...141

Figure 6.13. pE G F P _C 2_m S IG IR R C l o n i n g ...142

Figure 6.14. Unifying m e c h a n i s m ...143

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VII.

Table of Tables

T able 1.1. M arkers o f m icroglial a c tiv a tio n ... 4

T able 1.2. C ytokine classification and f u n c tio n ... 7

T able 1.3. Interleukin-1 R eceptor/T oll-like R eceptor S u p erfam ily ... 12

Table 1.4. R eceptors for A m y lo id -p ... 19

Table 2.1 M ouse PCR prim er assay n u m b e rs ... 46

Table 2.2 Sum m ary o f ELISA p ro to co ls... 47

Table 2.3 Sum m ary o f w estern im m unoblotting p ro to c o ls...48

Table 3.1. C av ea ts...53

Table 3.2. R esults s u m m a ry ... 58

Table 4.2. R esults s u m m a ry ... 83

Table 5.2. R esults s u m m a ry ...105

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1.1. Im m unity in the Central Nervous System (CNS)

The increasing prevalence o f neurodegenerative disorders because o f th e increase in the aging population has accelerated the efforts o f researchers to develop new therap eu tics aim ed at com bating the factors w hich contribute to the pathogenesis o f th ese disorders. M any neurodegenerative diseases, such as A lzh e im e r’s disease (A D ), P ark in so n ’s disease (P D ) and m ultiple sclerosis (M S), are associated w ith inflam m atory changes in the central nervous system (C N S). T his is thought to contribute to neuronal dam age and th erefo re m ay contribute to the progressive decline in cognitive fijnction. T herefore the in flam m atory response w hich is associated w ith glial activation and the secretion o f p ro-inflam m atory cytokines and chem okines by activated astrocytes and m icroglia has been targeted in the d ev elopm ent o f novel d rugs (S torer et al., 2005).

1.1.2. Astrocytes

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inflam m atory' response in the brain is thro u g h the production o f cytokines and chem okines. This cytokine-secreting capability is particularly pronounced w hen the C N S is in a diseased state. A strocytic production o f pro-in flam m ato ry cytokines and chem okines is considerably m ore pronounced in A D , PD and M S (B enveniste, 1998, H esseIgesser and H oruk, 1999, A kiyam a et a l , 2000). A strocytes are know n to be the prim ary source o f interleukin (lL )-6 and C hem okine (C -C m otif) ligand 2 (C C L 2). B oth m olecules play a significant role in the chem otaxis o f peripheral im m une cells to the C N S am ong o th er functions (see sections 1.3.2 and 1.3.4).

1.1 .3 . M ic r o g lia

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activating nearby m icroglia and o th er im m une cells and establishing a inflam m atory environm ent in the C N S (Perry, 2007, Soriano and Piva, 2008).

1.1.4. In flam m ation

The inflam m atory response is generally beneficial, neutralising the effects o f stim uli, infection or traum a. H ow ever dysregulation o f inflam m atory signaling is thought to cause or contribute to a wide range o f diseases. C ro h n ’s D isease and G uillain-B arre Syndrom e are know n to arise fi'om acute inflam m atory conditions. Persistence o f the inflam m atory response is associated w ith the developm ent o f chronic diseases such as rheum atoid arthritis. In addition, inflam m atory m ediators released from cancerous cells m ay contribute to m alignant progression o f tum ours (K ulbe et al., 2005). M ore recently, it has been recognised that inflam m atory conditions that arise secondary to m any neurodegenerative d iseases have been show n to contribute to the decline in cognitive function associated w ith neurodegenerative diseases (G riffin et al., 1995). A com m on link betw een these chronic conditions is the efficacy o f non-steroidal an ti-inflam m atory drugs (N S A lD s) in providing sym ptom atic re lie f to the patient, further indicating the role o f inflam m ation in th eir pathology (R ich et al., 1995, H arris et al., 1996, D eeks et al., 2002). The role o f chronic inflam m ation in neurodegenerative diseases is discussed in m ore detail in section

1.5. The im m une cells responsible for driving this inflam m ation e.g. m acrophages, dendritic cells and m icroglia, are activated through activation o f cell surface receptors w hich sense dam age. A cascade o f intracellular signaling often accom panies this process, w hich results in the upregulation o f inflam m atory m ediators e.g. cytokines, com plem ent proteins, and cluster o f d ifferentiation (C D ) m olecules.

1.1.5. LPS

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m e m b r a n e s o f g r a m - n e g a liv e bacterial cell w alls. It co n sists o f c o v a le n tly -b o u n d m o le c u le s o f

lipid and p o ly s a c c h a rid e . T L R 4 r e c o g n is e s L P S and interaction b e tw e e n the tw o in itiates an

intracellular s ig n a llin g c a s c a d e w h ic h resu lts in the u p re g u la tio n p ro - in f la m m a to r y gene

transcription. T h is p ro c e s s is d e sc rib e d in m o re detail belo w .

1.2. M a r k e r s o f m ic r o g lia l a c tiv a tio n

A n u m b e r o f p ro te in s are re c o g n is e d as m a r k e r s o f m icro g lia l a c tiv a tio n i.e. proteins th at sh o w a

c o n sisten t p a tte rn o f u p re g u la tio n in r e s p o n s e to stim uli k n o w n to cau se a c h an g e in the

activ atio n state o f m icroglia. A lth o u g h th e u p re g u la tio n o f th e se p ro tein s is indicative o f

functio n al c h a n g e s w ith in th e m ic ro g lia l cell, it is u n c le a r w h e th e r th e y p la y a direct role in these

functional ch a n g e s. T a b le 1.1 o u tlin e s a n u m b e r o f th e s e m ark ers. T h e m a rk e rs e x a m in e d in this thesis — C D l lb , C D 4 0 and C D 6 8 - a re d is c u s s e d in m o re detail below .

I'able 1.1. M a r k e r s o f m i c r o g lia l activ ation

M a r k e r F u n c tio n C e ll T y p e s

C D l lb C licm o tiix is; L euk (x ;v te a d h es io n

a n d m ig ra tio n .

M ic ro g lia , m o n o c ) ^

C D 4 0 r- c c ll re stiim ila tio n ; P h a g o c y to s is M ic ro g lia . m ac ro p h ag e s, a stro c y te s , en d o th e lia l cells C D 6 8 A n tig e n p re s e n ta tio n ; P h a g o c \1 o s is M ic ro g lia , m ac ro p h ag e s

C’!)8 6 C o -s tim u la tio n ; an tig en

p re sen ta tio n

M ic ro g lia , astro cy tes

C D 8 0 C o -s tim u la tio n ; an tig en

p re se n ta tio n

M ic ro g lia , m ac ro p h ag e s

M tlC il A n tig en p re se n ta tio n ; P h a g o c y to s is M ic ro g lia , m ac ro p h ag e s, astro cy tes

IC A M C'ell a d h e s io n M ic ro g lia , m ac ro p h ag c s.

le u k o c y tes, en d o th e lia l cells

C D 2 0 0 R M ic ro g lia l d e ac tiv a tio n M ic ro g lia . m ac ro p h ag e s,

m o n o cy te s

C X 3 C R I M ic ro g lia l d e a c tiv a tio n ; l- c e ll M ic ro g lia . m ac ro p h ag e s.

A d a p t e d fr o m L y n c h , 2 009.

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1.2.1. C D l l b

Integrins are a fam ily o f recep to rs w hich m ediate cell-cell interaction, cell adhesion to ex tracellu lar m atrix proteins and facilitate signalling pathw ays through their intracellular dom ains. T hey play key roles in developm ent, hom eostasis, im m une response and leukocyte trafficking. The latter characteristics have im plicated integrins in the d ev elopm ent o f a u to im m une diseases. C D l lb is a m em ber o f the integrin fam ily w hich is co n stitutively expressed on m onocytes, granulocytes, natural killer cells, m acrophages and m icroglia (H ynes, 2002, Solovjov et al., 2005). C D l lb can bind to CD 18 to form the tw o subunits m aking up integrin aiviPa, w hich is involved in the adhesion and m igration o f im m une cells. C D l l b expression is m arkedly increased in m icroglia w hen the cells are in an active state and upregulated in response to LPS and am yloid-P (A P) (P erera et al., 2001, S eabrook et al., 2006). In addition, C D l l b is upregulated in brain tissue in m any disease m odels including A P P /P S l tran sg en ic m ouse m odels o f A D and experim ental autoim m une encephalom yelitis (E A E ), a m odel for M S (A gnello et al., 2002, Y an et al., 2003).

1.2.2. CD40

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1.2.3. C D 68

C D 68 is a 1 lOkD, heavily glycosylated, m ucin-like m em brane protein found on endosom al and cell-surface m em branes o f m acrophages and m icroglia. It shares significant sequence hom ology o f the m em brane proxim al and cytoplasm ic dom ains w ith a fam ily o f lysosom al proteins including LA M P-1 (H olness and Sim m ons, 1993). C D 68 is thought to play a role sim ilar to M H CII in antigen presentation and m ay also m aintain the integrity o f the lysosom al m em brane (K urushim a et al., 2000). D igestion o f m aterials phagocytosed by cells occurs in the lysosome, and the upregulation o f C D 68 on the surface o f the lysosom e is indicative o f lysosom al, and hence phagocytic, activity.

1.3. C ytok in es

The p ro-inflam m atory cytokines upregulated upon activation o f m icroglia and astrocytes exist in low concentrations in the healthy brain, w here th ey play im portant roles in cell-cell signalling. H ow ever under conditions o f neurological stress th eir expression is increased and m ay lead to

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T a b l e 1.2. C y t o k i n e classification a n d function

C lassification

P r o - i n f l a m i n a t o r )

A n t i - i n l l a m m a t o r y

C X C C h e m o k i n e s

('C' C h e m o k i n e s

C C h e m o k i n e s C X ', C C h e m o k i n e s

I m n iu n o r e g u l a t o r y

C 'o i o n ) - s t im u l a t i n g factors

A n g i o g e n i c / 11 b ro g e n i c

I ' N t S u p c r llim il\

O t h e r

C ytokine

I L - l a . l l . - i p . IL-6. IL-7, IL -1 6 . I L -1 7 . IL -18

11,-lRA. 11.-4. II.-IO. I C i l ' - p i . I GI -P2 . I L - l i ' 5 . 1 L -IF 6

C X C L I , C X C 1 .2 , C X C L 3 , C X C L 4 , C X C L 5 , C X C I . 6 , C X C L 8 . C X C I . 9 . C X C I . I O CC1.2. C C I .3 . C C L 4 . C C M 7 . CC 1.18. C C L I 9 . CC1.20. C C L 2 2 X C l . l . X C L 2

C X j C l . l

l l ' N a , IFNy. I1.-12. IL-23

C}-CSF. M-C'SF. ( i M - C S l - . 11.3.

SCI-l SCI-l B - SCI-l i G r . I l G t . l GI -2 . I GI a . VFXjF

T N F a . H A I F . C D 3 0 I . . C I ) 9 3 L . I. r p . R A N K L

H D N F . N G F . A m p h i r e g u l i n . o n c o s ta tin . P B E F

Function

P r o m o t io n o f s y s te m ic in f la m m a tio n

S u p p r e s s io n o f s y s te m ic in lla m m a tio n

C h e m o t a x i s o f n e u tr o p h ils , l y m p h o c y t e s

C 'h e m o ta x is o f m o n o c y t e s . N K cells, d e n d ritic cells a n d I'-cells C h e m o t a x i s o f T -cells

C h e m o t a x i s o f T-cells. m o n o c y te s; le u k o c y te a d hes io n

I m m u n e cell a ctivatio n; 'F-cell ditTerentiation

I m m u n e cell proliferation a n d d ifferen tiatio n

A n g i o g e n c s is ; F ib r o g e n e s is Cell

g r o u t h , p ro lif eratio n .

diO'erentiation

Initiation o f a p o p to tic cell d e ath

1.3.1. IL -lp

I L - ip is b elieved to be secreted prim arily by m icroglial c e ll. T w o stim uli com b in e to initiate the a ssem b ly o f inflam m asom es. T h ese m ulti-protein co m p le x e s contain a proteolytic en zym e, ca sp a se-1 , w h ich c le a v e s p r o -lL - ip into its solu b le active product I L -ip (B asu et al., 2 0 0 4 ). In the p resen ce o f neurotoxic stim uli, such as w ould occur fo llo w in g brain trauma or in n eurodegenerative d isea ses, m icroglia upregulate the syn th esis o f I L -ip w here it contributes to

an exaggerated inflam m atory response. The I L -ip receptor has been localised on m icroglial c ell

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1.3.2. IL-6

IL-6 o p erates prim arily as a pro-inflam m atory cytokine in the C N S, w ith some reported an ti inflam m atory effects (Pais et a l , 2008). Like IL - ip , IL-6 is secreted from both activated m icroglia and astrocytes, w ith the latter seem ing to bear responsibility for the m ajority o f lL -6 production (V an W agoner et al., 1999). LPS has been identified as a potent stim ulator o f IL-6 from glial cells, and sim ilar effects w ere seen in glia cultured from brains o f aged rats (Xie et al., 2003). T he upregulation o f IL-6 is accom panied by an am plification o f the inflam m atory response. A lthough lL -6 itse lf contributes to this directly through its role as a pro-inflam m atory cytokine, it also exacerbates neuroinflam m ation further through its actions on other cells i.e. astrocytes (X ie et al., 2003). B inding o f lL -6 to it’s receptor leads to phosphorylation o f two kinases that associate w ith the receptor, Janus kinase (JA K )l and JAK2. These kinases subsequently activate STA T-1 and STA T3 w hich can initiate transcription o f pro-inflam m atory genes by tran slo catin g to the nucleus (V an W agoner and B enveniste, 1999). This pathw ay is know n as the JA K -S T A T signaling pathw ay and it is the prim ary route by w hich lL -6 signaling is transduced. Increased concentrations o f IL-6 in the C N S have been reported in several neurodegenerative diseases including A D and PD (M ogi et al., 1996, B aranow ska-B ik et al., 2008).

L3.3. TNF-a

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o f T N F R l-a sso c ia te d death dom ain protein (T R A D D ) to the death dom ain o f the T N F R trim er. The binding o f T R A D D to T N F R m ay facilitate several signaling pathw ays w ith vary in g end results (W ajant et al., 2003). These include stim ulation o f the m itogen activated protein kinase (M A R K ) pathw ay w hich m ay lead to cell proliferation, pro-ap o p to tic pathw ays that result in cell death and the N F -kB pathw ay w hich, as described earlier, results in the u p regulation o f pro-

inflam m atory genes. M icroglial-derived T N F -a has been im plicated in cell d eath associated w ith neurodegenerative diseases including A D and PD (T w eedie et al., 2007).

1.3.4. CCL2

CCL2 is classed as a chem okine due to its sm all size, four conserved cysteine residues and ability to facilitate chem otaxis o f nearby cells. A lthough C C L 2 can be secreted by neurons and astrocytes, the prim ary source o f the chem okine in the C N S are m icroglial cells (R a n so h o ff et al., 1993, Ishizuka et al., 1997) although both astrocytes and m icroglia release C C L 2 w hen exposed to LPS (Ling et al., 2010). Increased concentrations o f C C L 2 have also been reported to be associated w ith certain neurodegenerative disorders and anim al disease m odels, including AD and EA E (R a n so h o ff et al., 1993, G odiska et al., 1995, Ishizuka et al., 1997). In these cases, C C L 2 w as found to recruit m acrophages and T -cells into the C N S. P ro-inflam m atory cytokines i.e. I L - ip , IL-6 and T N F -a are thought to induce the release o f C C L 2, w hich in turn m ay act on nearby cells to facilitate the release o f m ore pro-inflam m atory cyto k in es in a positive feedback cycle (C o ta et al., 2000, R ankine et al., 2006).

1.3.5. CXCLIO

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et al., 1998). Increased concentrations o f C X C LIO have also been reported in the C N S in EA E and in brains from patients w ith m ultiple sclerosis (R an so h o ff et al., 1993, G erard and Rollins, 2 0 0 1).

1.3.6. C C L 3

C CL3 is a chem okine know n for its ability to selectively prom ote the m igration o f neutrophils and o th er granulocytes. It has also been show n to prom ote the upregulation o f pro-inflam m atory cytokine genes (K arpus and K ennedy, 1997). C onstitutive expression o f this chem okine is low in resting m icroglia, how ever follow ing exposure to LPS, T N F -a or I L - ip the CCL3 gene is upregulated and there is increased secretion o f the protein (M cM anus et al., 1998). As w ith C C L 2 and C X C LIO , increased expression and secretion o f CCL3 has been associated with several disease m odels, including EA E and focal cerebral ischem ia (K arpus and K ennedy, 1997, T akam i et al., 1997).

1.4. P ro-in flam m atory signal transd u ction

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regulatory effect on interleukin-1/toll-like receptor signaling. It has em erged as a possible target for novel anti-inflam m atory therapies.

1.4.1. I n te r le u k in -l/to ll-lik e recep tor signalin g

M em bers o f the I L - l/T L R superfamily o f receptors are characterized by the presence o f a conserved sequence called the T o ll/I L -IR (TIR) domain. This domain, located in the cytosolic region, is thought to participate in hom otypic interactions with the T IR dom ains o f other receptors and accessory proteins. It is through such interactions that signaling cascades initiated by m em bers o f the I L - l/T L R superfamily are facilitated (Fitzgerald and O'Neill, 2000). The superfamily can be subdivided into two additional protein families based on their extracellular dom ains - the I L - l R l and T L R subgroups. M em ber o f these sub-families are represented in F igu re 1.1. The extracellular dom ains o f m em bers o f the I L - l R l subgroup are distinguishable by the presence o f im munoglobulin (Ig)-like domains, w hereas TLR s typically consist o f leucine-rich repeats. The I L - l R l subgroup takes its name from the first protein in the I L - l/T L R superfamily to be described: the type 1 receptor, I L - l R l . In its role as receptor for the ubiquitous pro-inflam matory cytokine I L - ip , I L - l R l initiates signaling cascades that lead to the phosphorylation o f the transcription factor N F -k B and the M APKs, p38 and JN K (O'Neill and Dinarello, 2000). The toll subgroup com prises 13 m em bers ( T L R l- 1 3 ) which are expressed in humans. TLR 4, a receptor found on the surface o f immune cells and is the most studied m em ber o f the family. The signaling cascades that follow LPS-induced activation o f T LR 4 are similar to those associated with interaction between I L - i p - I L - l R l and result in phosphorylation o fN F -K B and the M A P K s (C how et al., 1999). This signaling cascade is illustrated in Figure 1.2. Certain accessory proteins thus have the ability to interact with either activated IL-1R1 or TLR4, so both receptors em ploy c o m m o n signaling pathw ays in response to different stimuli (O'Neill and Dinarello, 2000). The end biological effect o f this signaling pathw ay is the upregulation o f genes that encode for pro-inflam matory molecules. The I L - IR /T L R superfamily is outlined in T able

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T a b le 1.3. In te rle u k in -1 R e c e p to r/T o ll-lik e R e c e p to r S u p e rfa m ily

R ecep tor Function L igand(s)

I L - lR l C y to k in e re c e p to r; p ro - in fla m m a to ry sig n a l tra n s d u c tio n

IL-1

l l.- lR A c P i’ro -in lla m m ato r> sig n al tra n s d u c tio n

IL-1

I L - lR r p 2 I^ ro -in flam m ato ry sig n a l tra n s d u c tio n

i L - i r 6

1L-18R I’ro -in fla m m a to ry sig n al tra n s d u c tio n

IL -1 8

11.-RAPL i^ ro -in n a m m a to ry sig n a l tra n s d u c tio n

N /A

S IG IR R I)eco> re c e p to r N /A

I'lG lR R D e co y re c e p to r N /A

I L RI F o rm s d im e r w ith TiJ-12; p ro - P e p tid o g ly c a n . triac>l in fla m m a to ry sig n al

tra n s d u c tio n

lip o p ro te in s , zy m o san

T L R 2 Forn>s d im e r w ith T1>R1; p ro - P e p tid o g ly c a n . lip o te ic h o ic in fla m m a to ry • sig n al a c id , lip o p ro te in . tra n s d u c tio n lip o a ra b in o m a n n a n , zy m o s an ,

a m y lo id -p ri.R 3 P ro -in fla m m a to ry sig n al

tra n s d u c tio n

d s R N A

11.R4 F’ro -in fla m m a to ry sig n a l L P S , F p ro te in , h sp 6 0 . e th an o l. tra n s d u c tio n a m y lo id -p

H ,R 5 P ro - i n n am m at or_v sig n a l tra n s d u c tio n

I'lag c llin

T I,R 6 in te ra c ts w ith T L R 2 ; p ro - in fla m m a to ry sig n a l tra n s d u c tio n

l.ip o p ro te in

T L R 7 P ro -in fla m m a to ry sig n al tra n s d u c tio n

ss R N A , im iq u im o d

T L R 8 P ro -in fla m m a to ry sig n a l tra n s d u c tio n

ss R N A . im iq u im o d

'H .R 9 P ro -in fla m m a to ry sig n a l tra n s d u c tio n

L Jn m eth slated C'pG D N A

T L R IO P ro -in fla m m a to ry sig n a l tra n s d u c tio n

N /A

M Y D 8 8 U n iv e rsa l 1 LF^ a d a p to r N 'A M ai A d a p to r fo r T L R 2 , T L R 4 N /A

I'rif A d a p to r fo r T L R 3 N M

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TLR4 TLR2 I L - l R l SIGIRR

L e u c in e Rich R e p e a t s

Cell M e m b r a n e

T o ll / l n t e r l e u k i n - 1 R e c e p t o r D o m a i n

I

S e r4 4 7

Tyr536

I m m u n o g l o b u l i n D o m a in

Cys222 Leu3 05

F ig u re 1.1. T h e In te rle u k in -1 R e c e p to r/T o ll-L ik e R e c e p to r S u p e rfa m ily

[image:36.533.45.510.40.512.2]

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TLR4 IL-lRl

■' 'j " " '-: i L - ip 1 II--1RACP

iTiT»T^^'ylC^‘T'T

^M Al' ) J 2 MYD88 S

IBMl

UEMZJ

TRAF6

i TA K l

iKBa

N F -kB

F ig u r e 1.2. I L - 1 R 1 / T L R 4 sign alin g p a th w a y

[image:37.533.37.488.65.531.2]

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The I L - lR l and T L R signaling pathw ays play an integral ro le in the host response to infection. H ow ever, inappropriate o r excessive inflam m atory signaling m ay cause dam age to the host tissue and can often give rise to secondary diseases o r exacerbate an existing condition. A bnorm ally high concentrations o f IL-1 and o v er-expression o f gene products o f I L - lR l signaling have been reported in patients w ith inflam m atory diseases such as rheum atoid arthritis and G uillain-B arre syndrom e and have been im plicated in th eir pathogenesis (E astgate et al., 1988, S h arief et al., 1999). C hronic o ver-expression o f IL-1 P by activated m icroglia in the CN S

has been associated w ith accum ulation o f A p into plaques in neurons, and elevated concentrations o f the cytokine have been show n to exert a negative im pact on neuronal and synaptic function e.g., inhibiting spatial learning and long-term potentiation (L T P ) in the rat dentate gyrus (V erek er et al., 2000, M rak and G riffin, 2001). T his suggests that a persistent increase in I L - ip and AP signaling may be associated w ith the decline in cognitive function associated w ith AD.

G iven the num ber and severity o f diseases that may result from an excessive inflam m atory response, anti-in flam m ato ry drugs have long been prescribed for auto-im m une and auto-toxic diseases. N on-steroidal anti-in flam m ato ry drugs (N SA ID s) have proved effective in reducing the sym ptom s o f diseases such as rheum atoid arthritis. N S A ID s have a broad spectrum anti inflam m atory effect, prim arily achieved through cyclo-oxygenase inhibition. H ow ever, long term treatm ent o f chronic inflam m ation w ith N S A ID s is inadvisable due to reports o f side-effects in the kidney and gastrointestinal tract (W helton and H am ilton, 1991, Laine et al., 2003). It is now thought that m odulation, rather than suppression o f the inflam m atory response is a m ore appropriate m ethod o f treating inflam m atory diseases. T his has created a dem and for sm all m olecules w hich interact w ith and m odulate specific inflam m atory signaling pathw ays.

1.5. C hronic n eu roin flam m ation

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paralysis associated with Guillain-Barre syndrome, to potentially fata! allergic reactions. In the

context o f the CNS, however, chronic inflammation, that is, an inability o f C N S immune cells to

remove a stimulus, results in a persistently activated phenotype and a shift in the ratio o f pro-

inflammatory cytokines to anti-inflam m atory cytokines in the microenvironment. Chronic

inflammation can be induced by a num ber o f factors which m ay be related to changes in

physiological processes w ith aging, through the presence o f invading pathogens, chemical o r

physical insults or as a response to toxic peptide accumulation as occurs in some

neurodegenerative disorders.

1.5.1. A gin g and ch ron ic n eu roinflam niation

Aging is associated with significant neuronal loss and cognitive decline, and it is believed to be

the strongest risk factor for the developm ent o f neurodegenerative diseases (Lucin and W yss-

Coray, 2009). There is grow ing evidence to suggest that the negative impact o f aging on

cognitive processes is due to chronic inflammatory changes that lead to the loss o f neuronal

fibers (Licastro et al., 2005, Griffin et al., 2006). The major cause o f age-related chronic

inflammation is thought to be increased oxidative stress resulting from impaired anti-oxidative

m echanism s in cells which results in a build-up o f ROS over time (Chung et al., 2009). It has

been suggested that over time, mitochondrial D N A may become more susceptible to mutations,

which alter respiratory function and anti-oxidant capacity o f mitochondria leading to an

imbalance between the production and clearance o f ROS (Lin and Beal, 2006). Thus age-related

mitochondrial dysfunction can result in an accumulation o f ROS and ATP, both o f w hich are

capable o f activating intracellular signaling proteins such as stress-activated protein kinases and

N F -kB, although A T P is also recognised as a D A M P , inducing neuronal dam age by interacting

with P 2 X 7 R (Kim et al., 2002, M urp h y et al., 2012). ROS accumulation in the CNS can enhance

constitutive pro-inflam m atory signaling cascades in microglia and other im mune cells leading to

the production o f pro-inflam m atory cytokines such as IL-1(3, T N F - a and IL-6 and further

production o f ROS. These in turn are released from glial cells to act on neurons and other glial

cells to amplify pro-inflam m atory signaling cascades and contribute to neuronal damage and cell

death, and helping to maintain an inflammatory m icroenvironment within the CNS. While age-

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decline, it also has the effect o f m aintaining m icroglia in a hypersensitive, o r prim ed, state. There

is evidence to suggest that the prim ed m icroglia o f the aged brain exhibit increased expression o f

M H C ll and C D l l b , indicative o f activation, and exaggerated responses to p ro-inflam m atory

stim uli that m ay exacerbate inflam m ation and conseq u en t tissue dam age (B lock et al., 2007,

L yons et al., 2007a, C ox et al., 2012). This is one o f the reasons w hy age is the m ajor risk factor

for neurodegenerative d isorders driven by inflam m ation.

1.5.2. Neurodegenerative disease and chronic neuroinflam niation

N eurodegenerative diseases are characterised by the p rogressive loss, o r deterioration in

function, o f neurons and neurite outgrow ths in the peripheral nervous system (P N S ) and C N S.

T hese diseases m ay be induced o r exacerbated by environm ental factors but m any are also

associated w ith an underlying genetic m utation. A co m m on facto r in m any neurodegenerative

d isorders is the accum ulation o f toxic peptides, often m isfolded variants o f endogenously-

occurring proteins. O ne o f the m ost w ell characterised proteopathic neurodegenerative disorders

is C reutzfeld-Jakob D isease (C JD ), a condition in w hich native proteins are induced to m isfold

and accum ulate in toxic co ncentrations w ithin cells (N iim i et al., 2 008). T he result is progressive

cell loss and d eterioration in cognitive fiinction. M isfolded and aggregated toxic peptides have

been associated w ith o ther neurodegenerative diseases, including PD and AD. In these diseases,

protein aggregations may, in addition to directly inducing cell death, act as P A M Ps and stim ulate

a robust inflam m atory response w hich can exacerbate the effects o f the disease.

1.5.3. Parkinson’s Disease

PD is characterised by the profound loss o f dopam inergic neurons in the substantia nigra w hich

progressively im pairs m o to r ftinction and o th er form s o f cognition. O ne o f the factors associated

w ith the neuronal loss typical o f PD is the accum ulation o f a -sy n u c le in peptides into inclusions

know n as L ew y bodies, w hich can disrupt neuronal fijnctioning. D opam inergic neurons in the

PD brain are know n to release a variety o f factors, such as a -sy n u c le in and m atrix

m etalloproteinase 3 (M M P 3), w hich can induce the activation o f m icroglia and the release o f

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o f the disease (K im et a!., 2005, Z hang et al., 2005). A lthough the activated m icrogha accum ulate in large num bers in the substantia nigra in the PD brain, they are also present in a num ber o f o th er brain regions, including the hippocam pus (Im am ura et al., 2003). M ouse m odels in w hich a dopam inergic neurotoxin, 1-m ethyl 4-phenyl 1,2,3,6-tetrahydropyridine (M P T P ), is used to m im ic the neurodegeneration seen in PD, exhibit chronic m icroglial activation, and a sim ilar effect is seen in transgenic m odels o f a -sy n u c le in overexpression (H arvey et al., 2008, T heodore et al., 2008). It has also been reported that T N F -a and I L - ip as w ell as ROS, can contribute to neurodgeneration in PD (H irsch and H unot, 2009). It is w idely believed that the w idespread m icroglial activation and chronic inflam m ation typical o f PD contributes to the progression o f the disease and exacerbates m any o f its sym ptom s (L ong-S m ith et al., 2009).

1.5.4. A lzh eim e r’s d isease

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1.4. A(3 peptides also aggregate in and around neurons to form senile plaques which disrupt

synaptic function and attract activated microglia, ultimately leading to neurodegeneration

(Yankner et al., 1990, Qin et al., 2002). In vitro studies have dem onstrated the ability o f A p to induce the activation o f microglia and to promote the release o f pro-inflam m atory cytokines (Tan

et al., 2002, Lyons et al., 2007b, Jiao et al., 2008). It was also demonstrated that ICV injection o f

Ap induced an increase in the hippocampal expression o f M HCll and IL -ip (Lyons et al.,

2007b). M icroglia that accumulate around senile plaques have been shown to be immunopositive

for MHCH and CD68 (Akiyama et al., 2000, Gallagher et al., 2012). These studies demonstrate

the close relationship between microglial activation and AD, and highlight the importance o f

microglial activation as a target for treatm ents o f AD.

T able 1.4. R eceptors for Antyloid-P

R eceptor O th e r ligands

1 L R 2 l’e p l i d o g l > c a n . l i p o t e i c h o i c a c i d , l i p o p r o t e i n . 1 i p o a r a b i n o m a n n a n . z \ m o s a n

H, [< 4 L I ’S. I' p r o t e i n . h s p 6 0 . e t h a n o l

RAGi-; A G i ; . I I M C i B l . S lO O h . M a c - 1 , p h o s p h a t i d y l s e r i n e

C ' l ) 3 6 / C I ) 4 7 / a 6 p i - i n l c g r i n C o l l a g e n . th ro m h < ,) sp o n d in . 1.1)1,. l i p o p r o t e i n s , l a n i i n i n

S R - A l M o d l i l e d L D L

S R - B I I . D I , . I ID I .

a 7 - n i a ) t i n i c r e c e p t o r A c e t y l c h o l i n e , n i c o t i n e , a n a b a s i n e

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S o lu b l e A P P -p

A m y lo id P r e c u r s o r P r o t e i n (A PP)

A m y lo id -]

i - s e c r e t a s e

3 C ell M e m b r a n e

y - s e c r e t a s e

N i c a s t r i n A P H -1

P r e s e n i ! i n - 1 P E N -2

A P P c y to s o lic f r a g m e n t

F ig u r e 1.3. F o r m a ti o n o f am yloid -P p e p ti d e s

A p p e p tid e s are fo r m e d w h e n in teg ra l m e m b r a n e A P P is s e q u e n tia lly c le a v e d b y tr a n sm e m b r a n e p r o te o ly tic

e n z y m e s , y -s e c r e ta s e c le a v e s A P P in th e tr a n sm e m b r a n e r e g io n o f A P P w h ile P -s e c r e ta se , w h o s e a c t iv e s ite is

lo c a te d e x tr a c e llu la r ly , c le a v e s th e e x tr a c e llu la r p o rtio n o f A P P (B e n n e tt et a l., 2 0 0 3 , P o r te liu s et a l., 2 0 1 1 ) . T h e

p r o c e s s c a u s e s t h e r e le a s e o f s o lu b le A P P fr a g m e n ts a n d t o x ic A p p e p tid e s o f v a r y in g le n g th s . T h e m o s t c o m m o n

s p e c ie s o f A p p r o d u c e d b y th is p r o c e s s are c o m p o s e d o f 4 0 a n d 4 2 a m in o a c id r e s id u e s. A lth o u g h b o th s p e c ie s are

t o x ic to n e u r o n s a n d s tim u la te g lia l a c tiv a tio n , A P i^ 2 is m o r e fib r illo g e n ie a n d is th e r e fo r e m o r e a s s o c ia te d w ith th e fo r m a tio n o f p la q u e s se e n in A D . A third e n z y m e , a - s e c r e t a s e , is c a p a b le o f c le a v in g A P P c lo s e r to th e m e m b r a n e

than y -s e c r e ta s e a n d r e p r e se n ts a n o n -a m y lo id o g e n ic fo rm o f A P P p r o c e s s in g (Y in et a l., 2 0 0 7 , V a lle - D e lg a d o et a l.,

2 0 1 0 ) . E a r ly -o n se t A D h a s b een a ss o c ia te d w ith m u ta tio n s in th e g e n e s c o d in g for A P P a n d th e p r e s e n ilin -1

c o m p o n e n t o f y -s e c r e ta s e , illu str a tin g th e cen tra l r o le o f a m y lo id o g e n e s is in A D p a th o lo g y (M u r r e ll e t a l., 1 9 9 1 ,

[image:43.533.14.486.57.457.2]

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1.6. M od u lation o f inflam m atory signalling

It is evident that inflammatory signalling plays an important role in the response to insults to the

brain. It has also become clear that the dysregulation o f inflam m atory signalling associated with

age and neurodegenerative disorders can have a devastating impact on neuronal viability and

consequently on neuronal and synaptic function and therefore cognition. There are a num ber o f

endogenous factors which exert direct and indirect effects on inflam m atory cascades to either

prom ote or inhibit inflammatory signalling. C hanges in the expression o r function o f these

factors are a characteristic o f m any neurodegenerative disorders. The following section will

focus on a num ber o f proteins which exert a negative regulatory effect on inflam m atory

signalling, acting as a break on the inflammatory response.

1.6.1. F ractalkin e

Fractalkine is a chemokine which is expressed in a variety o f tissues and can exist in a

m em brane-bound o r soluble form (Bazan et al., 1997, Lyons et al., 2009). The m em brane-bound

form o f fractalkine is found in abundance on neurons while its receptor is highly expressed on

microglia (Harrison et al., 1998). This co m plem entary expression o f the chem okine and its

receptor supports a growing body o f evidence that describes a m odulatory role for fractalkine on

microglial activation. Exogenous application o f soluble fractalkine has been show n to attenuate

the LPS-induced increase in I L - l p from rat mixed glia and microglia. This effect was also seen

w hen glial cells w ere co-cultured with neurons, but the effect was lost follow'ing treatment with a

fractalkine blocking antibody, suggesting that the neuronal m em brane-bound form o f fractalkine

plays a similar role (Lyons et al., 2009). There is evidence w hich links this ability to modulate

cytokine release from glia to cognition, as fractalkine-deficient mice exhibit reduced learning

ability and LTP, which was reversed following application o f I L - l R a (R ogers et al., 2011).

Fractalkine deficiency has been shown to negatively impact the regeneration o f the olfactory

epithelium due to an inhibition in the proliferation o f progenitor cells and the increased

recruitment and activation o f macrophages (B lom ster et al., 2011). However, there is evidence o f

an alternative role for soluble fractalkine as a chemoattractant to peripheral im mune cells.

Increased fi-actalkine concentrations have been found in CSF o f MS patients and fractalkine

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2012). A num ber o f studies have investigated the role o f fractalkine in other neurodegenerative

diseases. There is conflicting evidence about its role in PD. Fractalkine concentrations in the

C SF o f PD patients appears to increase with the severity o f the disease but it is unclear whether

this is an indication o f a protective response o r due to the role o f fractalkine as a chemoattractant

for immune cells (Shi et al., 2011). Exogenously-applied fractalkine has shown differential

effects in different models o f PD, with microglial activation and dopaminergic neuron depletion

observed in an MPP* model whereas, in a 6-hydroxydopam ine (6-O H D A ) infusion model,

fractalkine suppressed microglial activation and reduced lesion size (Pabon et al., 2011, Shan et

a!., 2011). There have also been conflicting reports o f the role o f fractalkine in AD. Reduced

fractalkine concentrations have been observed in the plasma o f patients with severe A D

com pared with mild AD o r controls (Kim et al., 2008). Interestingly, fractalkine was also shown

to be reduced in the brains o f aged APPswe transgenic mice (Duan et al., 2008). Fractalkine-

deficient A P P -P S l mice have a profoundly altered C N S cytokine environm ent with reduced

m R N A expression o f T N F - a and CCL2 but increased I L - l p m R N A (Lee et al., 2010). It remains

unclear w hether fractalkine has a beneficial or detrimental role in PD and AD. It is possible that

the multiple functions o f the chemokine cause it to have differential effects as the disease

progresses, contributing to deleterious effects early on by promoting the migration o f peripheral

im mune cells w hich exacerbate the negative effects o f inflammation, but acting as an essential

m odulator o f microglia as the cells develop and becom e chronically over-active in later stages.

1.6.2. Cluster o f Differentiation 200 (CD200)

C D 200 is a transm em brane glycoprotein and m em ber o f the Ig superfamily o f proteins (W right

et al., 2000). It is widely distributed on a variety o f cell types, including neurons, and interacts

with its structurally similar receptor C D 200R w hich is located primarily on cells o f the myeloid

lineage (W right et al., 2003, Lyons et al., 2007a). C D 200 is thought to mediate in cell-cell

interactions and is an important m odulator o f myeloid cells, including microglia (Lyons et al.,

2007a). Evidence to date suggests that it is one o f the most important mechanisms o f neuronal

m odulation o f microglial function. C D 200 exerts its effects through inhibition o f the ras/M A P K

signalling pathway. C D 2 0 0 R binds to C D200 and is tyrosine phosphorylated leading to the

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D O K 2 are capable o f interacting w ith and activating ras G T P ase activating protein (R asG A P ),

w hich acts as an inhibitor o f ras by enhancing its G T P ase activity, inducing the no n -sig n allin g

G D P -bound form o f ras. T his u ltim ately reduces th e activ ity o f the transcription factors ERK,

JN K and p38 and dam pens dow n the p ro -in flam m ato ry gene expression th ey induce (Z hang et

a!., 2004).

Analysis of the impact of SIGIRR on glial function

Terms and Conditions of Use of Digitised Theses from Trinity College Library Dublin

Access Agreement

Analysis of the impact of SIGIRR on glial function

TRINITY C O L L E G E ^ 2 A MAY 2013

Declaration

Acknowledgements

Abbreviations

MAPK M itogen-activated protein kinase

M yD 88 M yeloid differentiation factor 88

PBS Phosphate buffered saline

Table of Contents

3 .3 .4 In v e stig a tin g th e e f f e c t o f I L - i p o n g lia l c e lls prepared from w ild ty p e and

3 .4 .7 . T h e e f f e c t o f I L - i p treatm en t o n m ic r o g lia from w ild ty p e and SIG IR R "' m i c e ...75

4 .4 .1 . O verview 94

Chapter 5: A n alysis o f the e ffect o f intrahippocam pal injection o f A p in you n g and m iddle-aged, w ildtype and S IG IR R -deficient m i c e ... 9 9 5.1. In tro d u ctio n ...100

5 .3 .5 . The e ffe c t o f age, A p and ab sen ce o f SIG IR R on N F -kB activation in cortical and hippocam pal t is s u e ...104

Table of Figures

Table of Tables

1.1. Im m unity in the Central Nervous System (CNS)

1.1.4. In flam m ation

1.2. M a r k e r s o f m ic r o g lia l a c tiv a tio n

I'able 1.1. M a r k e r s o f m i c r o g lia l activ ation

1.3. C ytok in es

C ytokine

1.3.5. CXCLIO

1.4. P ro-in flam m atory signal transd u ction

R ecep tor Function L igand(s)

TLR4 TLR2 I L - l R l SIGIRR

iTiT»T^^'ylC^‘T'T

1.5. C hronic n eu roin flam m ation

1.5.1. A gin g and ch ron ic n eu roinflam niation

1.5.2. Neurodegenerative disease and chronic neuroinflam niation

1.5.3. Parkinson’s Disease

1.5.4. A lzh eim e r’s d isease

A p p e p tid e s are fo r m e d w h e n in teg ra l m e m b r a n e A P P is s e q u e n tia lly c le a v e d b y tr a n sm e m b r a n e p r o te o ly tic

1.6.1. F ractalkin e

1.6.2. Cluster o f Differentiation 200 (CD200)