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Roles of microglial purinergic receptors in inflammatory conditions of the brain Choi, Hyun Beom 2006

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Roles of Microglial Purinergic Receptors in Inflammatory Conditions of the Brain by Hyun Beom Choi B.Sc. , Ajou University, 1998 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R OF P H I L O S O P H Y in T H E F A C U L T Y OF G R A D U A T E S T U D I E S (Experimental Medicine) T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A October 2006 © Hyun Beom Choi , 2006 A B S T R A C T Microglia , the resident immune cells of brain, mediate inflammatory responses leading to progressive neuronal damage in neurodegenerative diseases. Binding o f A T P to purinergic receptors activates microglia thereby inducing cellular responses in inflamed brain cells. The two families of purinergic receptors, labelled P 2 X R (ionotropic) and P 2 Y R (metabotropic) contribute to inflammatory responses in microglia. The first two parts of my study focused on the involvement and role of the ionotropic purinergic receptor, P2X7R in mediating inflammatory responses such as secretion of pro-inflammatory factors in vitro and in vivo. The final part of my study concentrated on purinergic receptor-dependent intracellular C a 2 + ([Ca 2 +]j) mobilization and functional responses in human fetal microglia. A detailed in vivo study was carried out on the involvement of P2X7R in mediating lipopolysaccharide (LPS)-induced inflammatory responses and neuronal damage in rat striatum. LPS-injected striatum exhibited a marked increase in the expression and production of P2X7R compared with control (saline)-injected animals. Additionally, L P S injection upregulated a host of pro-inflammatory mediators and reduced neuronal viability. The P 2 X 7 R antagonist, oxidized A T P (oxATP) was effective in attenuating expressions of all inflammatory mediators; most importantly o x A T P was protective for striatal neurons. In vitro, I found L P S stimulation of cultured human microglia enhanced cellular expressions of inflammatory mediators and increased [Ca 2 + ] , mobilization which were blocked with o x A T P treatment. Overall, the results from this work indicate that P 2 X 7 R plays a critical role in L P S -induced inflammatory responses including induction of neuronal damage. Subsequently a series o f studies was designed to examine putative roles of P2X7R in mediating inflammatory responses with relevance to the pathology typical of Alzheimer's disease (AD) . First, I found microglia isolated from A D brains expressed enhanced P 2 X 7 R i i compared with microglia obtained from non-demented individuals. In a second study, human fetal microglia stimulated with AP1-42 peptide exhibited markedly elevated levels o f P2X?R compared with untreated cells. Also, P2X7R-mediated C a 2 + responses were increased with Api .42 pretreatment of cells relative to untreated cells. Finally, in vivo double immunostaining analysis showed considerable P2X7R co-localized with microglia following injection of APi_ 42 into rat hippocampus. The overall results from this section of study show the involvement of P2X7R in mediating microglial purinergic inflammatory responses in A D brain. We were also interested in the contribution of purinergic receptors other than P2X7R in mediating inflammatory responses. I focused on cyclooxygenase-2 (COX-2) since this enzyme is highly elevated in inflamed brain and contributes to inflammation-induced cytotoxicity. In this research, we used a low concentration of A T P (100 uM) to eliminate contributions o f P2X7R since activation of this purinergic subtype receptor requires concentrations of A T P in excess of I m M . In summary, we found that the block of P 2 X R (candidate P2X4R) increased the duration of ATP-mediated [Ca ]< responses and upregulated expression and production of C O X - 2 . The prolonged response involved influx of C a through store-operated channels (SOC) and was suggested as a consequence of removal of cell depolarization by the block of P 2 X R . Inhibition of S O C was then shown to be effective in attenuating C O X - 2 expression in human microglia. These novel results link inhibition of P 2 X R , other than P 2 X 7 R , with upregulation of C O X - 2 in human microglia with the link involving SOC-mediated C a 2 + influx. The overall findings from this study suggest that pharmacological manipulation of P2X7R and other purinergic receptors could serve as a potential therapeutic intervention in modulating inflammatory responses in microglia. 111 T A B L E O F C O N T E N T S A B S T R A C T i i T A B L E O F C O N T E N T S iv L I S T O F T A B L E S . v i i L I S T OF F I G U R E S : v i i i L I S T OF A B B R E V I A T I O N S x A C K N O W L E D G E M E N T S x i i i D E D I C A T I O N xiv S T A T E M E N T OF C O N T R I B U T I O N S xv C H A P T E R 1: G E N E R A L I N T R O D U C T I O N . 1 1.1 M I C R O G L I A : G E N E R A L 1 1.1.1 Properties of Microgl ia in Inflammation ; 4 1.1.1.1 Pro-inflammatory Mediators , 6 1.1.1.2 Pro-inflammatory Cytokines 6 1.1.1.3 Reactive Oxygen Species (ROS) 8 1.1.1.4 Cyclooxygenase-2 (COX-2) 9 1.1.1.5 Anti-inflammatory Mediators 10 1.1.2 Microglia in Neurodegenerative Disease 11 1.1.2.1 Microgl ia and A D '. 12 1.1.2.2 Microgl ia and P D 13 1.1.2.3 Microglia and H D 14 1.1.2.4 Microglia and M S 15 1.1.2.5 Microgl ia and A L S 15 1.2 P U R I N E R G I C R E C E P T O R S 17 1.2.1 P 2 Y receptors (P2YR) in C N S 17 1.2.1.1 Topology o f P 2 Y R 20 1.2.2 P 2 X receptors (P2XR) 20 1.2.2.1 Topology of P 2 X R 22 1.2.2.2 Subtypes of P 2 X R in C N S 24 1.2.2.3 Heteromeric P 2 X R 25 1.3 P U R I N E R G I C R E C E P T O R S IN M I C R O G L I A 27 1.3.1 Purinergic P 2 Y R and P 2 X R (not P2X 7R)-mediated [Ca 2 + ] , Mobilization 27 1.3.2 Store-operated Channels (SOC) in Microgl ia 28 1.3.3 P2X 7R-mediated Signal Transduction Pathways 37 1.4 R E S E A R C H H Y P O T H E S I S '. 40 1.5 S U M M A R Y OF P R O P O S E D R E S E A R C H O B J E C T I V E S . 41 1.6 R E F E R E N C E S 42 iv C H A P T E R 2: M O D U L A T I O N O F T H E P U R I N E R G I C P 2 X 7 R E C E P T O R A T T E N U A T E S L P S - M E D I A T E D M I C R O G L I A L A C T I V A T I O N A N D N E U R O N A L D A M A G E 54 2.1 I N T R O D U C T I O N ; 54 2.2 M A T E R I A L S A N D M E T H O D S f 57 2.2.1 Animals 57 2.2.2 Cel l Culture : 57 2.2.3 Immunohistochemistry 58 2.2.4 R T - P C R 59 2.2.5 C a 2 + Spectrofluorometry 62 2.2.6 Quantitative Analysis 63 2.2.7 Statistics and Analysis 63 2.3 R E S U L T S .' 64 2.3.1 Effects of L P S on P 2 X 7 R Expression and Production in Rat Brain 64 2.3.2 Co-localization of P 2 X 7 R with Gl ia l Cells in Rat Brain... 66 2.3.3 Effects of o x A T P on LPS-mediated Microgl ia l Activation 68 2.3.4 Effects o f o x A T P on LPS-induced Activation of p38 M A P K and N F K B 70 2.3.5 Effects of o x A T P on LPS-induced i N O S Expression. 74 2.3.6 Effects of o x A T P on LPS-mediated Nitration 77 2.3.7 Effects of o x A T P on LPS-mediated L ip id Peroxidation and Oxidative D N A Damage 80 2.3.8 Effects of o x A T P on LPS-induced Neuronal Caspase-3 Activation 83 2.3.9 Effects of o x A T P on LPS-induced Neuronal Loss 85 2.3.10 Effects of o x A T P on LPS-induced Gene Expressions in Cultured Human Microgl ia 87 2.3.11 Effects of o x A T P on LPS-modulated [Ca 2 +]j in Cultured Human Microglia . . . . 91 2.4 D I S C U S S I O N 94 2.5 R E F E R E N C E S 101 C H A P T E R 3: U P R E G U L A T E D E X P R E S S I O N O F P U R I N E R G I C P 2 X 7 R E C E P T O R I N A L Z H E I M E R ' S D I S E A S E A N D A M Y L O I D - B E T A P E P T I D E T R E A T E D M I C R O G L I A A N D I N P E P T I D E I N J E C T E D R A T H I P P O C A M P U S 107 3.1 I N T R O D U C T I O N 107 3.2 M A T E R I A L S A N D M E T H O D S 110 3.2.1 Adult Human Microgl ia 110 3.2.2 Fetal Human Microgl ia 111 3.2.3 R T - P C R Analysis I l l 3.2.4 Preparation and in vitro and in vivo Application of Amylo id Beta Peptide 113 3.2.5 Calcium Sensitive Fluorescence Microscopy 114 3.2.6 Immunohistochemical Analysis 114 v 3.2.7 Statistical Analysis 115 3.3 R E S U L T S 116 3.3.1 P2X7R Expression in Microglia from A D Patients and N D Individuals 116 3.3.2 P 2 X 7 R Expression in APi.4 2-treated and Untreated Human Microgl ia 116 3.3.3 Functional Responses of P2X7R in AP1.42 Pretreated and Untreated Human Microgl ia 119 3.3.4 P 2 X 7 R Expression in Rat Hippocampus 122 3.4 D I S C U S S I O N 126 3.5 R E F E R E N C E S 129 C H A P T E R 4: D I F F E R E N T I A L A C T I V A T I O N OF P U R I N E R G I C R E C E P T O R S M O D U L A T E S L E V E L S OF COX-2 A N D [ C A 2 + ] i I N H U M A N M I C R O G L I A 131 4.1 I N T R O D U C T I O N : 131 4.2 M A T E R I A L S A N D M E T H O D S 133 4.2.1 Preparation and Culture of Human Microgl ia 133 4.2.2 Calcium Spectrofluorometry 133 4.2.3 Reverse-Transcription P C R (RT-PCR) 134 4.2.4 Immunocytochemistry 135 4.2.5 Cellular Morphology and Viabil i ty 136 4.2.6 Solutions and Reagents 136 4.3 R E S U L T S 138 4.3.1 Characterization of [Ca 2 + ]i Responses by Purinergic Receptor Modulators 138 4.3.2 Effects of Purinergic Receptor Modulators on COX-2 m R N A Expression 144 4.3.3 P 2 X R Modulate COX-2 Protein Expression 147 4.3.4 Effects of Purinergic Agents on Morphology of Microgl ia 150 4.4 D I S C U S S I O N 152 4.5 R E F E R E N C E S 156 C H A P T E R 5: C O N C L U S I O N S OF THESIS R E S E A R C H A N D F U T U R E D I R E C T I O N S 160 v i LIST O F T A B L E S Table 1-1. Compounds inducing microglial activation : 3 Table 1-2. Compounds produced by microglia '. 5 Table 1-3. Agonists and antagonists of the P 2 Y R subtypes and signal transduction mechanisms 19 Table 1-4. Agonists and antagonists of the P 2 X R subtypes and signal transduction mechanisms.... 23 Table 2-1. Gene specific P C R primer sequences 61 v i i LIST O F FIGURES Figure 1-1. Topology of purinergic receptors 21 Figure 1-2. A T P (applied at 100 uM)-induced changes in [Ca 2 +]j in human microglia.. . 31 Figure 1-3. Expression of subtype of P 2 X R in human microglia 34 Figure 1-4. Simplified schematic diagram of purinergic-mediated C a mobilization in human microglia 36 Figure 2-1. L P S induces expression and production of P 2 X 7 R in rat striatum 65 Figure 2-2. P 2 X 7 R is co-localized with microglia and astrocytes in rat brain 67 Figure 2-3. O x A T P reduces the number of activated microglia in L P S (5 |o,g for 3 d) injected rat brain 69 Figure 2-4. O x A T P inhibits activation of p38 M A P K and N F K B (p65 subunit) in L P S -injected rat striatum 72 Figure 2-5. O x A T P attenuates LPS-mediated i N O S expression in rat striatum 76 Figure 2-6. O x A T P reduces LPS-mediated nitration of proteins 79 Figure 2-7. O x A T P reduces LPS-induced lipid peroxidation and oxidative D N A damage .' .' 82 Figure 2-8. Neuronal caspase-3 is activated in a time-dependent manner and is inhibited by blocking P 2 X 7 R ; ...84 Figure 2-9. O x A T P increases neuronal viability in LPS-injected rat brain (3 d post-LPS injection) 86 Figure 2-10. O x A T P blocks LPS-induced pro-inflammatory mediators, but not anti-inflammatory cytokines in cultured human microglia 89 Figure 2-11. L P S pretreatment modulates P 2 X 7 R mediated [Ca 2 + ] i mobilization in cultured human microglia 93 Figure 2-12. Schematic diagram depicting a putative P2X 7 R-mediated scheme linking neuronal damage to L P S stimulation 100 Figure 3-1. Expression of P 2 X 7 R i n N D and A D brain and in A(3[.42 stimulated human fetal microglia 118 Figure 3-2. Changes in [Ca ]i induced by B z A T P 121 Figure 3-3. In vivo expression and levels of P 2 X 7 R in rat hippocampus 124 Figure 3-4. Single and double immunostaining for P 2 X 7 R 125 9-1-Figure 4-1. Modulation of A T P induced changes in [Ca ]j 140 Vll l Figure 4-2. [Ca 2 +]j responses elicited by A T P plus P P A D S and with B z A T P 142 Figure 4-3. R T - P C R for purinergic modulation of C O X - 2 expression in treated human microglia 146 Figure 4-4. Purinergic modulation of C O X - 2 production in human microglia 148 Figure 4-5. Morphological changes in human microglia following treatments 151 ix LIST O F ABBREVIATIONS A A arachidonic acid A(3 amyloid beta peptide A p , . 4 2 42-residue C-terminal variant of amyloid beta peptide A042-1 42-residue C-terminal variant of reverse amyloid beta peptide A C adenylyl cyclase A D Alzheimer's disease A D P adenosine diphosphate ADP-p -S adenosine diphosphate-p-S 2 - A G 2-arachidonoylglycerol A L S amyotrophic lateral sclerosis A P P amyloid precursor protein A T P adenosine 5' triphosphate B B B blood-brain barrier B z A T P 2X3>0-(4-benzoylbenzoyl) A T P °C celsius degree [Ca 2 + ] i intracellular calcium concentration Caspases cysteine aspartyl proteases c D N A complementary deoxyribonucleic acid C N S central nervous system C N T F ciliary neurotrophic factor C O X - 1 / 2 cyclooxygenase-112 C P A cyclopiazonic acid C s A cyclosporin A D A P I 4,6-diaminodino-2-phenylindole D G L diacrylglycerol lipase D M E M Dulbecco' s modified Eagle' s medium E A A excitatory amino acid E A E experimental autoimmune encephalomyelitis E G T A ethylene glycol-bis (2-aminoethyl-ether)-A^,A^,A^',Af'-tetraacetic acid E R endoplasmic reticulum E R K - 1 / 2 extracellular signal-regulated kinases-1/2 Fura -2 /AM fura-2 acetoxymethylester G A P D H glyceraldehyde-3 -phosphate dehydrogenase G F A P glial fibrillary acidic protein, astrocyte cell marker G F P green fluorescent protein G M - C S F granulocyte macrophage-colony stimulating factor H D Huntington's disease HEK293 human embryonic kidney cell line H E P E S A^-(2-hydroxyethyl)piperazine-A^-(2-ethanesulfonic acid) 4 - H N E 4-hydroxynonenal I C A M - 1 intracellular adhesion molecule-1 icv intracerebroventricular IFN-y interferon gamma IL-4 interleukin-4 X IL-10 interleukin-10 IL-12 interleukin-12 I L - l p interleukin-ip IL-6 interleukin-6 IL-8 interleukin-8 i N O S nitric oxide synthase i.p. intraperitoneal IP 3 inositol triphosphate Kca Ca 2 +-dependent outward K + channel LIF leukemia inhibitory factor L P S lipopolysaccharide LY294002 PI3-K inhibitor M - C S F macrophage-colony stimulating factor M C P - 1 macrophage chemoattractant protein-1 M G L monoacylglycerol lipase M H C major histocompatibility complex M I P - W - i p macrophage inflammatory protein-1 a / - ip M P T P l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine M S multiple sclerosis NeuN neuronal cell marker N D non-demented N F K B nuclear factor kappa B N O nitric oxide 3-NT 3-nitrotyrosine 8-OHdG 8-hydroxy-2'-deoxyguanosine O x A T P oxidized A T P OX-42 microglial/macrophage cell marker P A F platelet activating factor P B R peripheral benzodiazepine receptor P B S phosphate buffered saline P D Parkinson's disease PD98059 E R K - 1 / 2 inhibitor P F A paraformaldehyde PGs prostaglandins PI3-K phosphatidylinositol 3-Kinase PK11195 1 -(2-chlorophenyl)-N-methyl-N-( 1 -methylpropyl)-3 isoquinolinecarboxamide P L A 2 / C / D phospholipase A 2 / C / D p38 M A P K p38 mitogen-activated protein kinase P P A D S pyridoxal-phosphate-6-azophenyl-2\ 4"-disulfonic acid OPSS C a 2 + free physiological saline solution PSS physiological saline solution P T X pertussis toxin P 2 X R ionotropic purinergic receptors P 2 X 7 R P 2 X R subtype P 2 Y R metabotropic purinergic receptors R N S reactive nitrogen species R O C receptor-operated channels R O S reactive oxygen species R T - P C R reverse transcriptase polymerase chain reaction SB203580 p38 M A P K inhibitor S E M standard error of the mean S E R C A sarcoplasmic/endoplasmic reticulum Ca 2 + -ATPase SKF96365 store-operated channels inhibitor S O C store-operated channels TGFp-1 tumor growth factor (3-1 T N F - a tumor necrosis factor-a TNFR1/2 tumor necrosis factor-a receptor 1/2 U D P uridine diphosphate U T P uridine triphosphate V C A M - 1 vascular cell adhesion molecule-1 A C K N O W L E D G E M E N T S I would like to express my foremost gratitude to my supervisors, Dr. James G . McLarnon and Dr. Seung U . K i m for their precious guidance throughout my graduate study. Without their patience and support, this work would not have been completed. Their teaching wi l l be with me throughout my career and has inspired me to follow in their footsteps. I am also grateful to my committee members, Dr. Gary Quamme and Dr. Peter Soja, for their assistance and invaluable insights into this work. I would like to acknowledge the following: Jae K . Ryu and Dr. Sonia Franciosi for their enthusiasm, technical advice and suggestions. I also would like to express my gratitude to current and past lab members: Karen Tran, Wei Wei , Taesup Cho, Dr. Han S. Jeong. Dr. Atushi Nagai, Dr. Kozo Hatori, Dr. Seok H . Hong, Dr. Tae W . K i m , Vikram Goghari, Scott Hadland, Jeffrey Helm, Natinee Jarantonai, Prasongchai Sattayaprasert and Ladan Zand. In addition, I would like to acknowledge the Heart and Stroke Foundation of Canada (HSFC) for granting me a Doctoral Research Award (2004-2006). M y gratitude goes to Faculty, Staff and students in the Department of Anesthesiology, Pharmacology and Therapeutics and the Department of Experimental Medicine of U B C for their encouragement. v I would like to thank my parents for their unselfishness love and support throughout my life. Finally, I would like to thank my wife, Youn Hee and my two daughters, Y u Jin and Y u A h n for their patience and support. x i i i D E D I C A T I O N I would like to dedicate this thesis to my parents, my wife and two daughters. S T A T E M E N T O F CONTRIBUTIONS The chapters in this thesis contain work that has been previously published or submitted for publication in peer-reviewed journals. Choi H B , Ryu J K , K i m S U and McLarnon JG. Modulation of the purinergic P 2 X 7 receptor attenuates LPS-mediated microglial activation and neuronal damage. This study has been submitted to Journal o f Biological Chemistry (presented here as Chapter Two). McLarnon JG , R y u J K , Walker D G and Choi H B . Upregulated expression of purinergic P2X7 receptor in Alzheimer's disease and amyloid-beta peptide treated microglia and in peptide injected rat hippocampus. This study has been submitted to Journal o f Neuropathology and Experimental Neurology (presented here as Chapter Three). Choi H B , Hong S H , R y u J K , K i m S U and McLarnon J G . Differential activation o f subtype purinergic receptors modulates C a 2 + mobilization and C O X - 2 in human microglia. Gl ia 2003 43(2):95-103. (presented here as Chapter Four). The thesis author Hyun Beom Choi was the primary researcher for all the results presented in the articles above. Technical expertise with in vivo experiments was provided by Jae K . Ryu and adult microglia from Alzheimer's diasease ( A D ) patients and non-demented (ND) individuals were provided by Drs. Douglas G . Walker and Lih-Fen Lue from Sun Health Research Institute (Arizona, U S A ) . We agree with the stated contributions of the thesis author, as stated above. Dr. James G . McLarnon (thesis co-supervisor) Dr. Seung U . K i m (thesis co-supervisor) xv C H A P T E R 1: G E N E R A L INTRODUCTION 1.1 M I C R O G L I A : G E N E R A L Microglia , the immune cells of the central nervous system (CNS), represent approximately 10-20% of the glial cell population. These cells were first identified and characterized in 1932 (del Rio-Hortega, 1932). Immunocytochemical studies using macrophage-specific markers have suggested that microglia originate from monocytes entering the C N S at early stages of embryonic development (Perry et al., 1985; L ing and Wong, 1993). Other possibilities have been considered such as microglia being derived from mesenchymal progenitor cells or that they are of neuroectodermal origin with cells originating from glioblasts or the germinal matrix (Kaur et al., 2001). Microgl ia are distributed throughout the C N S and have two distinct morphologies; ameboid and ramified. The latter is indicative of a resting state showing a small cell body with thin and branched processes and the former is indicative of an activated state showing a roundish cell body with attenuated processes. Resting microglia act as immune surveillance cells in the C N S participating in mechanisms of innate and adaptive immunity. Recent studies using time-lapse two-photon imaging of green fluorescent protein (GFP)-labeled microglia have demonstrated that microglial processes are highly dynamic in the intact brain (Davalos et al., 2005; Nimmerjahn et al., 2005). Upon activation, microglia undergo not only a morphological shift from ramified to ameboid but also upregulation of a variety of cell surface markers. Activation involves complement receptor 3 (CR3) and a host of receptors mediating microglial cellular responses. Activated microglia also express major histocompatibility complex ( M H C ) class I and II which have a role in presenting antigen to T lymphocytes. 1 Microgl ia respond to a host of stimuli such as adenosine triphosphate (ATP) , platelet-activating factor (PAF) and lipopolysaccharide (LPS). Compounds activating microglia are listed in Table 1-1. A body of evidence suggests that chronic activation of microglia leads to cellular production of neurotoxic substances (Giulian et al., 1995; Righi et al., 1995; Wang et al., 2000). Factors secreted by microglia include pro-inflammatory cytokines, reactive oxygen species (ROS), proteases, arachidonic acid derivatives, excitatory amino acids ( E A A ) and quinolinic acid (see section 1.1.1). These factors have been implicated in contributing to pathology in neurodegenerative diseases such as Alzheimer's disease (AD) , Parkinson's disease (PD), amyotropic lateral sclerosis ( A L S ) and multiple sclerosis (MS) (McGeer et al., 1988; Tooyama et al., 1990; Aimer et al., 2001; Copelman et al., 2001; Lue et al., 2001; Gao et al., 2002). Agents released from damaged or endangered neurons drive microglia into a gradual transformation stage and many of the secretory products of activated microglia such as pro-inflammatory cytokines and chemokines are autocrine, leading to chronic activation of these cells and recruitment of more microglia to the site of injury. Astrocytes are also an important source for signaling factors which alter functional responses of microglia. Overall a number of paracrine and autocrine processes contribute to activation of microglia and the generation of inflammatory responses. Since inflammation contributes to the progressive loss of neurons in neurodegenerative diseases, understanding the underlying complex mechanisms of inflammation serves as an important step in developing useful neuroprotective pharmacological agents. 2 Table 1-1. Compounds inducing microglial activation Class of compound Stimuli Antibodies Complement Cytokines Chemokines Endogenous proteins Ions Neurotrophic factors Neurotransmitters Other proteins Serum factors and proteases Surface molecules of infectious agents Other compounds IgA, IgG, IgM C l q , C5a IL-1P, IL-2, IL-4, IL-6, IL-10, IL-12, IL-15, IL-18, IFN-y, TGF-p, TNF-a, GM-CSF, M-CSF M l P - l a , RANTES, GROp, PF4, Fractalkine, IL-8 A p , prion protein (PrP) K + , M n 2 + BDNF, GDNF, NGF, NT-3, NT-4 1 P-adrenergic agonists, ATP and related purines, glutamate, kainate, N M D A ApoE, CD40L, M S H , endothelin, S100B, VIP Albumin, Thrombin LPS, bacterial cell wall proteoglycans, LTA, gp41, gpl20 Cannabinoids, ceramide, gangliosides, LPA, melatonine, opiods/endomorphines, PAF, PGE2, steroids Abbreviations: IgA, G , M : immunoglobulin A , G , M ; M l P - l a : macrophage inflammatory protein 1-a; R A N T E S : regulated upon Activation, Normal T-cell Expressed and Secreted; PF4: platelet factor 4; B D N F : brain-derived neurotrophic factor; G D N F : glial-derived neurotrophic factor; N G F : nerve growth factor; N T - 3 , -4 neurotrophin-3, 4; N M D A : N-methyl-D-asparate; A p o E : Apolipoprotein E ; M S H melanocyte stimulating hormone; V I P : vasoactive intestinal peptide; L P S lipopolysaccharide; L T A : lipoteichoic acid; gp41, 120: glycoprotein 41, 120; L P A lysophosphatidic acid; P A F : platelet activating factor; P G E 2 : prostaglandine E 2 . 3 1.1.1 Properties of Microglia in Inflammation A number of reviews have considered the roles of microglia in mediating inflammatory responses in brain (McGeer and McGeer, 1995; Kreutzberg, 1996; Gonzalez-Scarano and Baltuch, 1999; Alo i s i , 2001). Microgl ia are the resident immune cells of the brain and perform a variety of functions integral to the operation of the brain under physiological and pathological conditions. In the former case, microglia can secrete neurotrophic factors and anti-inflammatory agents which promote healing and enhance neuronal viability. However, microglia are readily stimulated by changes in homeostatic conditions and the presence of foreign invaders such as abnormal proteins and surface structures of infectious agents. Activated microglia exhibit-proliferative responses and production of toxic factors (Kreutzberg, 1996; Giulian, 1999). In extreme cases of chronic inflammation, microglial responses may actually amplify and exacerbate the effects of brain stimuli leading to excessive damage to neighboring cells such as neurons. A number of inflammatory mediators with neurotoxic potential produced by microglia. A list of compounds produced by microglia are included in Table 1-2. A t present, it seems that none of these factors acting alone is implicated as an overall causative mediator of neuronal death. Rather, available data suggest that an overall aggregation of inflammatory mediators can lead to progressive ki l l ing of neurons. Microgl ia express a large repertoire of signaling pathways. Receptor-mediated responses are recorded for stimuli such as L P S , amyloid peptide (A(3), cytokines, chemokines, A T P and P A F . Indeed, for a given stimulus such as A p , multiple types of receptors may be activated (Bamberger et al., 2003). 4 Table 1-2. Compounds produced by microglia Compound Function ATP and related purines B7-1,B7-2,CD40 BDNF, bFGF, Plasminogen HGF, NGF, NT-3, , C l q , C3, C4 COX-2 Glutamate, Quinolinic acid I L - l a , I L - l p 1L-3 IL-4, IL-10, TGF-p IL-6 IL-12 IL-1RA IL-8, M I P - l a , M I P - i p , MCP-1, RANTES MMP-2, MMP-9 PAF PGE2 02", NO, ONOO", OH" Chemoattractant Co-stimulatory factor of antigen presentation Neurotrophic factors Complements Pro-inflammatory molecule Excitotoxins Pro-inflammatory cytokines Hematopoietic growth factor Anti-inflammatory cytokines Pro/Anti-inflammatory cytokines Promotion ofThl cells Anti-inflammatory compound Chemoattractants Matrix metalloproteinases Bioactive lipid, neurotoxic Anti-inflammatory molecule Oxidative stress Abbreviations: B D N F : brain-derived neurotrophic factor; b F G F : basic fibroblast growth factor; H G F : hepatocyte growth factor; N G F : nerve growth factor; N T - 3 : neurotrophin-3; C O X - 2 : cyclooxygenase-2; M I P - l a : macrophage inflammatory protein 1-a; M I P - i p : macrophage inflammatory protein 1-P; M C P - 1 : monocyte chemoattractant protein-1; R A N T E S : regulated upon Activation, Normal T-cell Expressed and Secreted; P A F : platelet activating factor; P G E 2 : prostaglandine E 2 . 1 5 Following receptor binding, numerous downstream processes are activated to modulate, signals including mobilization of C a 2 + , gating of ion channels, and activation of G -proteins, tyrosine kinases, M A P kinases, transcription factors and others. The resultant microglial response to a stimulus is then shaped by a complexity of interacting pathways and largely dependent on the nature of the stimulus. A t present, however, the precise coupling mechanism(s) which link a particular activating stimulus with a specific cellular functional process such as proliferation, phagocytosis and secretion of inflammatory mediators are not well understood. 1.1.1.1 Pro-inflammatory Mediators Activated microglia secrete a host of substances such as interleukins ( I L - i p , IL-6), tumor necrosis factor alpha (TNF-a) , R O S and cyclooxygenase-2 (COX-2) which are responsible for a variety of cellular and molecular signaling pathways in the C N S . The roles of pro-inflammatory mediators in microglia are briefly discussed below. 1.1.1.2 Pro-inflammatory Cytokines I L - i p : I L - i p is a potent pro-inflammatory signaling molecule normally expressed at low level; the cytokine is induced rapidly in response to local or peripheral stimuli. IL-1P is implicated in the production of a number of other pro-inflammatory mediators such as IL-6 and in transformation of diffuse amyloid deposits into neuritic A p plaques (Griffin et al., 1995) and spread of amyloid plaques in A D brains (Sheng et al., 1995). It has been also reported that stimulation of microglia by I L - i p enhances expression 6 and production of macrophage inflammatory protein-la ( M l P - l a ) , M I P - l p and T N F -a (Chao et al., 1995; McManus et al., 1998). I L - i p has been implicated in increased acetylcholinesterase (AChE) activity leading to decreased acetylcholine (ACh) levels in the brain resulting in cognitive decline (L i et al., 2000). Reduced A C h has been reported in A D patients (Wynn and Cummings, 2004). Microgl ia express I L - i p converting enzyme (ICE), the enzyme responsible for converting pro-IL- ip into its mature active form which may contribute to the inflammatory response after C N S injury or inflammation (Eriksson et al., 1999). I C E overexpression from microglia in the vicinity of plaques can lead to neuronal degeneration characterized by neuritic A p plaque progression and neurofibrillary tangles in Alzheimer's disease (Sheng et al., 1998; Griffin and Mrak, 2002). Interestingly, overexpression of I L - i p is associated with excessive tau phosphorylation in vivo and in vitro (Sheng et al., 2001; L i et al., 2003). IL-6: IL-6 exhibits both pro- and anti-inflammatory effects in brain (Raivich et al., 1999). IL-6 shares the signaling receptor gpl30 with other growth factors including ciliary neurotrophic factor (CNTF) , leukemia inhibitory factor (LIF) and IL-11. During brain inflammation IL-6 released from activated microglia or infiltrated macrophages inhibits the production of new neurons in adult brain. In in vitro studies, neurogenesis is reduced in the presence of activated microglia, but not resting (quiescent) microglia (Monje et al., 2003). Unlike the other pro-inflammatory cytokines, IL-6 does not induce multiple pro-inflammatory genes. A s noted above, some evidence suggests that IL-6 may also exhibit anti-inflammatory actions. A s one example, IL-6 can inhibit glial production of T N F - a induced by IFN-y, I L - i p and the bacterial endotoxin L P S (Benveniste, 1992). Overall 7 findings indicate that IL-6 exerts predominant pro-inflammatory responses in inflamed and aged brain. T N F - a : T N F - a is one of the major cytokines that is produced by activated myeloid lineage cells and microglia are the major source of T N F - a in the C N S . T N F - a has two distinct receptors, the p55 T N F receptor ( T N F R 1) and the p75 T N F receptor ( T N F R 2). The latter has a trophic or protective role in neuronal survival (Antel et al., 1996), however, the former plays a role in cell death when activated (McKee et al., 1998). Both neurotoxic and neuroprotective effects of T N F - a have been reported. T N F - a has been documented to k i l l human cortical neurons (Mogi et al., 1994), whereas the cytokine has been reported to be neurotrophic to rat hippocampal neurons and protect enriched cultures of primary neurons from glutamate, free radical and A p toxicity (Barger et al., 1995; Barger and Harmon, 1997; Akiyama et al., 2000). In terms of intracellular pathways T N F - a stimulates N F K B , a transcription factor which is associated with production of other pro-inflammatory factors. T N F - a has shown to be elevated in A D brain and has been shown to increase the production of A p (Blasko et al., 1999). 1.1.1.3 Reactive Oxygen Species (ROS) Oxidative stress is an imbalance between cytosolic free radicals and the protective capacity of cells. A growing body of evidence suggests that oxidative stress plays an important role in the pathophysiology of neurodegeneration (Beal, 1992; Mark et al., 1996). Microgl ia are the major cells in the C N S that secrete reactive oxygen species (ROS) and reactive nitrogen species in response to inflammatory stimuli. 8 Microglial production of superoxide anions (O2") and nitric oxide (NO) leads to the formation of the cytotoxic compound, peroxynitrite ( O N O O ) (Pryor and Squadrito, 1995). Peroxinitrite can diffuse over considerable distance causing oxidative damage in reacting with proteins, lipids and D N A (Stamler et al., 1992; Beckman and Crow, 1993; Pryor and Squadrito, 1995). Neuroprotective effects have been documented in L P S inflammation models by inhibition of inducible nitric oxide synthase (iNOS) and nicotiamide adenine dinucleotide phosphate ( N A D P H ) oxidase. These are key enzymes for N O and superoxide anion production, respectively (Gao et al., 2002; Arimoto and Bing, 2003). The nitration of tyrosine residues (indicated by 3-nitrotyrosine) on proteins, lipids and D N A serve as a marker for peroxynitrite-mediated oxidative stress (Zhang et al., 2000). L ip id peroxidation is an autocatalytic process commenced by R O S generated by cellular oxidative stress. 4-Hydroxynonenal (4-HNE) is a major end product of l ipid peroxidation and used as a marker for l ipid peroxidation (Ejima et al., 2000). The level of 4 - H N E is relatively high in some neurodegenerative diseases such as Alzheimer's disease (AD) and cerebral ischemia (McGrath et al., 2001; McKracken et al., 2001). Previous work from this laboratory has reported blockade of excitotoxin-induced lipid peroxidation (4-HNE) and oxidative D N A damage (8-OHdG) by inhibiting microglial activation (Ryu et al., 2005). 1.1.1.4 Cyclooxygenase-2 (COX-2) . C O X - 2 is the key enzyme for converting arachidonic acid ( A A ) to prostaglandins (PGs). The latter are critical mediators of physiological and inflammatory processes 9 produced by activated microglia during brain inflammation. C O X is known to exist as two isoforms: C O X - 1 is constitutively expressed by many cell types and is responsible for homeostatic production of prostanoids and C O X - 2 is inducible at the site of inflammation. A growing body of evidence indicates that C O X - 2 levels are increased in A D brain and correlate with the levels of A p (Kitamura et al., 1999). Non-steroidal anti-inflammatory drugs (NSAIDs) exert anti-inflammatory effects through the inhibition of C O X - 2 enzymatic activity thereby preventing P G synthesis. N S A I D s reduce microglial activation following infusion of A p peptide in rats and the number of activated microglia in non-demented elderly arthritic individuals. Selective inhibition of C O X - 2 prevents glial activation and neuronal cell death in an animal model of A D (Giovannini et al., 2002). It should be noted that C O X - 2 inhibitors can also act on neuronal C O X - 2 . Indeed, neurons are one of the major cell types expressing this enzyme (Kunz and Ol iw, 2001; Kawaguchi et al., 2005). Overall, inhibition of C O X - 2 enzymatic activity appears beneficial in neurodegenerative disease. 1.1.1.5 Anti-inflammatory Mediators A host of factors known as anti-inflammatory mediators are secreted from microglia and provide immunosuppressive and neuroprotective effects in the C N S . Chronic inflammatory progression is one of the pathophysiological mechanisms in neurodegenerative diseases such as A D (Akiyama et al., 2000). Thus, anti-inflammatory intervention is a major research topic in neurodegenerative disorders. The roles of anti-inflammatory mediators in microglia are briefly discussed below. 10 TGF-P: Transforming growth factor beta (TGF-P) exerts potent immunosuppressive actions on microglia stimulated by granulocyte macrophage-colony stimulating factor ( G M - C S F ) or macrophage-colony stimulating factor (M-CSF) (Suzumura et al., 1993). TGF-P inhibits the production of pro-inflammatory mediators such as T N F - a , IL-1 , IL-6, expression of M H C class II and proliferation of microglia. This anti-inflammatory cytokine also inhibits the formation of superoxide anion by L P S stimulated microglia. Mice lacking TGF-P demonstrated elevated levels of M H C class II with the incidence o f fatal multifocal inflammatory disease strikingly high (Shull et al., 1992). T G F - p has also been reported to block cytokine induction of endothelial adhesion molecules such as intracellular adhesion molecule-1 ( ICAM-1) and vascular cell adhesion molecule-1 ( V C A M - 1 ) . The consequence is a reduced activation of T cells and extravasation of inflammatory cells into brain parenchyma (Shrikant et al., 1996; Winkler and Benveniste, 1998). IL-10: IL-10 is a typical anti-inflammatory cytokine rendering immunosuppressive and neuroprotective actions in the C N S . IL-10 has been reported to inhibit the production of pro-inflammatory mediators including I L - i p , IL-6, IL-12, T N F - a , G M - C S F and M - C S F in monocytes (Wang et al., 1995). Systemic overexpression of IL-10 has been reported protective against experimental autoimmune encephalomyelitis ( E A E ) , an animal model of multiple sclerosis (MS) (Bettelli et al., 1998). IL-10-deficient mice showed an increased severity of E A E (Falcone et al., 1998). 1.1.2 Microglia in Neurodegenerative Disease Detailed reviews are available concerning roles of inflammatory responses in the pathology of specific diseases: Alzheimer's disease ( A D ; McGeer and McGeer, 1995; 11 Akiyama et al., 2000), Parkinson's disease (PD; Gao et al, 2003b), Huntington's disease (HD; Tabrizi te al., 1999), multiple sclerosis ( M S ; Benveniste, 1997) and amyotrophic lateral sclerosis ( A L S ; McGeer and McGeer, 2002). The following is a brief summary for roles of microglia in neurodegenerative disease. 1.1.2.1 Microglia and Alzheimer's Disease Alzheimer's disease (AD) is a progressive brain disorder and is characterized by gradual cognitive decline and memory loss. A D is the most common form of dementia and the incidence and prevalence of A D increases considerably with age (Kawas et al., 2000). Pathological hallmarks of A D are aggregation of amyloid beta peptide (AP) and neurofibrillary tangles, particularly in the hippocampus and cerebral cortex (Selkoe, 1999). Activated microglia are located in areas of A D pathology and are associated with dystrophic neurites and highly insoluble Ap deposits and neurofibrillary tangles (Rogers et al., 1988; Akiyama et al., 2000). These microenvironmental changes in the brain could lead to hyperactivation of microglia and release of inflammatory factors and neurotoxins contributing to neuronal damage and cognitive decline in A D (Giulian, 1999; Akiyama et al., 2000). The continued presence of Ap deposits may sustain microglial activation resulting in chronic inflammation in the brain (Cotman et al., 1996; Ryu et al., 2004). A t present, the mechanisms involved in modulating cell signaling in stimulated microglia are not well understood; however, the treatment of rodent microglia or human monocyte T H P cells with Ap has been found to induce changes in tyrosine kinase and mitogen-activated protein kinase signaling pathways (McDonald et al., 1997,1998; Combs et al., 1999). 12 Altered signal transduction in A D microglia could potentially be damaging to neurons (Banati et al., 1993; Dickson et al., 1993; Akiyama et al., 2000). Recent work from our laboratory has shown that stimulus-induced Ca 2 +-dependent signaling properties of human microglia are perturbed in microglia isolated from A D patient's brain relative to microglia obtained from non-demented (ND) individuals. Since microglia serve a critical role in mediating inflammatory responses, our findings may have particular relevance to neurodegeneration in A D . Furthermore, we also find that Afi\^2 treatment of human fetal microglia alters Ca 2 +-dependent pathways in a manner similar to that found for A D microglia (McLarnon et al., 2005). We also reported that a significantly increased expression of P 2 X 7 R in microglia from A D patients relative to N D individuals and in microglia exposed to APi„ 4 2 compared with untreated cells. It has been also reported that aggregated A p applied to microglia from both N D and A D individuals induces dose-dependent increases in production of pro-inflammatory cytokines, chemokines, and several other inflammatory factors (Lue et al., 2001). Microgl ia from A D patients and Ap-stimulated microglia from N D individuals secreted significantly higher levels of complement component C l q , R N S and macrophage-colony stimulating factor (M-CSF) (Lue et al., 2001). 1.1.2.2 Microglia and Parkinson's Disease P D is characterized by a slow and progressive loss of dopamine-producing neurons in the substantia nigra. A recent review has detailed the utility in using different anti-inflammatory maneuvers to attenuate damage to nigrostriatal neurons in animal models of P D (Gao et al, 2003a). Previously, a number of studies have implicated 13 inflammatory responses mediated by microglia in contributing to neuronal damage in P D (Vi la et al., 2001; Gao et al., 2002; W u et al., 2002). Particular factors which have been suggested as causative in damage to dopaminergic neurons include superoxide (Liu et al., 2000; Gao et al., 2002), nitric oxide (Liberatore et al., 1999; L i u et al., 2002) and pro-inflammatory cytokines (Grunblatt et al., 2000). Reactive free radicals and their downstream products have been shown to contribute substantially to the oxidative damage in P D . Inhibition of N A D P H oxidase or reduction in N O production by activated microglia reduced neuronal degeneration induced by L P S , rotenone, l-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or in combination of L P S and rotenone (Liberatore et al., 1999; W u et al., 2002; Gao et al., 2003b). Specific agents with anti-inflammatory actions have been found to protect neurons in PD-relevant conditions including minocycline (Wu et al., 2002), naloxone (Liu et al., 2000) and C O X - 2 inhibitors (Teismann and Ferger, 2001). In a M P T P mouse model of P D , a reduced microglia activation was correlated with enhanced neuroprotection for dopaminergic neurons (Breidert et al., 2002). 1.1.2.3 Microglia and Huntington's Disease Elevated levels of oxidative damage products and activated glial cells are found in the brains of H D patients (Browne et al., 1999; Sapp et al., 2001). Degeneration of striatal neurons in H D has been associated with actions of reactive oxygen and nitrogen species (Tabrizi et al., 1999). The presence of abnormal huntingtin protein could provide a potent stimulus for the aggregation and activation of microglia in H D brain (Sapp et al, 2001). Excitotoxic injury has been implicated as a causative factor in H D and is associated with 14 chronic activation of microglia (Mitchell et al., 1993). Our laboratory has found inhibition of microglia to be associated with increased neuronal survival in an excitotoxic animal model of H D (Ryu et al., 2005). A t present, however, the mechanism(s) by which inflammation leads to neuronal damage is not well understood. 1.1.2.4 Microglia and Multiple Sclerosis M S lesions exhibit extensive myelin damage and are characterized by chronic inflammatory responses due to high expressions of infiltrating T cells (Hickey, 1999). Microgl ia could induce damage by releasing pro-inflammatory cytokines, matrix metalloproteinases ( M M P s ) and free radicals, which are markedly elevated in M S brain (Benveniste, 1997; Sriram and Rodriguez, 1997). Recent work has implicated microglial involvement in M S lesions, including induction of C O X - 2 (Rose et al., 2004) and expression of M H C Class I (Hoftberger et al., 2004). In an animal model of M S , experimental autoimmune encephalomyelitis (EAE) , the involvement of CD8+ microglia/macrophages in the pathogenesis of demyelination has been reported (Schroeter et al., 2003). 1.1.2.5 Microglia and Amyotropic Lateral Sclerosis Neuroinflammation in A L S brains is characterized by extensive microgliosis in areas of degenerating neurons and in upregulation of activated microglia markers (Lampson et al., 1990; Kawamata et al., 1992). Increased expressions and levels of C O X -2 have been found in A L S spinal cord relative to unaffected regions (Yasojima et al., 2001), however, the cellular source of the C O X - 2 was not identified. The involvement of 15 oxidative stress as a factor in A L S has also been considered (Cleveland and Rothstein, 2001; Shibata et al., 2001), however, antioxidant therapy has generally not proven effective in the treatment of this disease (Louwerse et al., 1995; Vyth et al., 1996). The regulation of microglial respiratory burst activity in addition to the use of antioxidants has been suggested as a measure to inhibit oxidative stress in A L S (McGeer and McGeer, 2002). These investigators also indicate that control of other mediators from activated microglia could be used to inhibit inflammatory responses in A L S . 16 1.2 PURINERGIC R E C E P T O R S The purinergic receptors are known to respond to extracellular nucleotides including ATP, A D P , U T P and U D P and mediate physiological responses. The purinergic receptors are composed of two families labelled as PI and P2 receptors. The PI receptors are most specifically sensitive to adenosine and have 4 subtypes designated as A i , A 2 A , A 2 B and A 3 . These receptor subtypes play important roles in normal cell physiology including activation of phospholipase C (PLC) and adenylyl cyclase (AC) and modulation of ion channels. The PI receptors w i l l not be discussed further since my research focuses on the role of P2 receptors in microglia. Two P2 receptor families have been classified and designated as P 2 X receptors (P2XR) and P 2 Y receptors (P2YR) . The latter are G-protein coupled receptors whereas the former receptors are directly linked to cationic ion channels (Dubyak and El-Moatassim, 1993; Burnstock, 1996). Microgl ia can express both P 2 X R and P 2 Y R . The former is associated with influx of N a + and C a 2 + and efflux of K + through non-selective cationic channels leading to cellular depolarization and the latter causes intracellular Ca 2 + ( [Ca 2 + ] i ) mobilization. A more detailed summary of properties of P 2 X R and P 2 Y R is included below. 1.2.1 P2Y receptors (P2YR) in CNS P 2 Y R are termed metabotropic receptors and are comprised o f P2Y]R , P2Y2R, P 2 Y 4 R , P 2 Y 6 R , P 2 Y n R , P 2 Y , 2 R , P 2 Y 1 3 R a n d P 2 Y i 4 R . P 2 Y R are G-protein coupled and linked to inositol 1,4,5-triphosphate (IP3)-mediated release of C a 2 + from endoplasmic reticulum (ER) stores. Depletion of C a 2 + from E R stores can subsequently trigger entry of C a 2 + through a plasmalemmal store-operated channel (SOC) reported in numerous types 17 of nonexcitable cells (Hoth and Penner, 1992). A t present, the roles of SOC in shaping and modifying functional microglial processes are not well characterized. Recombinant P 2 Y R activate phospholipase C (PLC) or A C . The former induces release of IP3 and the latter alters cyclic adenosine monophosphate ( c A M P ) levels. However, endogenous P 2 Y R activate much more complex intracellular signaling pathways including phospholipase A 2 , C and D ( P L A 2 , P L C , P L D ) , mitogen activated protein kinase ( M A P K ) , extracellular signal-regulated kinases-1/2 (ERK-1/2) , tyrosine kinase and A C . Agonists and antagonists for the P 2 Y R subtypes and signal transduction mechanisms are summarized in Table 1-3. It should be. noted that many purinergic antagonists exhibit non-selective effects with actions on different purinergic receptors dependent on concentration. 18 Table 1-3. Agonists and antagonists of the P2YR subtypes and signal transduction mechanisms Subtype Agonist Antagonist G protein Effector P2Y,R A D P > ATP RB-2,MRS2179,PPADS G„„ J ^ O R O C K P 2 Y 2 R ATP=UTP Suramin Gq/n + G, P2Y 4 R uxP = A T P « h ) P P A D S Gq/n + G, tPLC tPLC TPLC P2Y 6 R UDP>UTP>ADP RB-2, Suramin, PPADS G q / 1 1 , P2Y„R ATP Suramin, RB-2 ^ q / " P 2 Y 1 2 R ADP > ATP ^ S A M T ' ^ ^ ^ ^ ' G' U C , T P D K P2Y, 3 R ADP > ATP MRS2211 G; l l c a ( N ) Abbreviations: h: human; r: rat; P L C : phospholipase C; A C : adenylnyl cyclase; R O C K : Rho-dependent kinase; I K i r : current mediated by inwardly rectifying potassium channels; I C A ( N ) : current mediated by N-type voltage-dependent calcium channels; I K ( M ) : M-type potassium current; PI3K: phosphoinositide 3-kinases ; N / D : not determined; M R S 2211: pyridoxal-5'-phosphate-6-azo-(2-chloro-5-nitrophenyl)-2,4-disulfonate; A R - C 6 7 0 8 5 M X : 2-(propylthio)-P,y-dichloromethylene-ATP; R B - 2 : reactive blue-2; P P A D S , pyridoxal-5'-phosphate-6-azo-phenyl-2,4-disulfonate; CI330-7: Nl-(6-Ethoxy-l,3-benzothiazol-2-yl-2- (7-ethoxy-4-hydroxy-2,2-dioxo-2H-2-6benzo[4,5][l,3]thiazolo[2,3-c][l,2,4]thiadiazin-3- yl)-2-oxo-l-ethanesulfonamide; M R S 2179: 2'-Deoxy-N6-methyladenosine-3',5'-bisphosphate; 2 - M e S A M P : 2-Methylthioadenosine-5'-monophosphate. 19 1.2.1.1 Topology of P2YR The topology of metabotropic P 2 Y R is characterized by an extracellular N -terminus and an intracellular C-terminus; the latter has consensus binding motifs for protein kinases. P 2 Y R subtypes have a diverse structure of intracellular loops and C -terminus which influences coupling with one or more G-protein subtypes. Mammalian P 2 Y R comprise 328 to 370 amino acids and have 7 transmembrane (TM) spanning regions, which help to form the ligarid docking compartment. The T M 3 , 6 and 7 regions have particularly high sequence homology. However, overall sequence homology at the peptide level among P 2 Y R subtypes is relatively low (19-55% homology) leading to pharmacological diversity among subtypes. A representative topology of a P 2 Y R subunit is presented in Figure 1-1 A . 1.2.2 P2X receptors (P2XR) The ionotropic P 2 X R family is directly coupled to non-selective cationic channels allowing influx of N a + and C a 2 + and efflux of K + . To date, seven distinct P 2 X R subtypes have been identified at the molecular level and named P 2 X i R - P 2 X 7 R . A n important consequence of activation of P 2 X R is cell depolarization, which can serve as a modulatory factor in shaping cellular responses. The P 2 X 7 R is a unique family member and is described in considerable detail in section 1.3.3; It is also a focus of study in my experimental work. 20 P2YR P2XR C O O H C O O H Figure 1-1. Topology of purinergic receptors A : Left diagram illustrates P 2 Y R subtypes. Extracellular N-terminus and intracellular C -terminus. The latter has consensus binding motifs for protein kinase. B: Right diagram represents P 2 X R subtypes. N - and C - termini are intracellularly located. A large extracellular domain contains multiple conserved cysteine residues. C I and C2 represent conserved multiple cysteine residues forming disulphide bonds. 21 1.2.2.1 Topology of P2XR The hydrophobicity plots of the P 2 X R subunits suggest the presence of only two hydrophobic transmembrane domains instead of three or four present in other ionotropic receptor families (Hollmann et al., 1994; Methot et al., 1994; Valera et al., 1994; North, 1996). N - and C - termini are intracellularly located and extracellular region is present between the two transmembrane domains. Mammalian P 2 X R monomers are 339 to 595 amino acids long and the presence of multiple motifs for N-l inked glycosylation in the extracellular domain are reported (Torres et al., 1998). A large extracellular domain contains 10 highly conserved cysteine residues forming disulphide bonds (North, 2002). A representative topology of a P 2 X R subunit is presented in Figure 1-1B. Most channels are oligomeric in order to form a pore large enough to pass cations. The stoichiometry for the P 2 X R has not been firmly established, however, initial studies using functional assay of biochemical cross-linking and concatameric receptors suggest that these receptors are composed of either trimers or multiples of trimers (Nicke et al., 1998; Stoop et al., 1999). ATP, a purinergic ligand is a putative signaling molecule from damaged neurons serving as a chemotactic signal for the proliferation and activation of microglia. The lists of agonists and antagonists for the P 2 X R subtypes and signal transduction mechanisms are summarized in Table 1-4. 22 Table 1-4. Agonists and antagonists of the P2XR subtypes and signal transduction mechanisms Subtype Agonist Antagonist Effector P2X!R a,p-meATP, HT-AMP, IsoPPADS, MRS2159, Cation channel PAPET-ATP NF203,Suramin p (Ca27Na+) ~4 P2X 2 R 2-MeSATP, ATPyS, A p 4 A RB-2, Suramin Cation channel , p (Ca 2 +/Na +) ~2 P2X 3 R a,P-meATP, HT-AMP, IsoPPADS, NF203, Cation channel PAPET-ATP TNP-ATP p (Ca 2 +/Na +) ~4 P2X 4 R 2-MeSATP, CTP B B G Cation channel p (Ca 2 +/Na +) ~4 P2X 5 R 2-MeSATP, dATP Suramin Cation channel P2X 6 R N / A N / A N / A P 2 X 7 R BzATP Ox-ATP, KN-62, B B G Cation channel, Pore formation Abbreviations: a ,p-meATP: a,P-methylene adenosine-5'-triphosphate; H T - A M P : 2-Hexylthioadenosine 5'-monophosphate; P A P E T - A T P : 2-[2-(4-Aminophenyl)ethylthio]adenosine 5'-triphosphate; P A P E T - A T P : 2-[2-(4-Aminophenyl)ethylthio] adenosine 5'-triphosphate; A T P y S : Adenosine 5'-0-(3-thiotriphosphate); 2 - M e S A T P : 2-Methylthioadenosine 5'-triphosphate; A p 4 A : Diadenosine tetraphosphate; C T P : Cytidine 5'-triphosphate; d A T P : 2'-Deoxyadenosine 5' triphosphate; B z A T P : 3'-Benzoylbenzoyl adenosine 5'-triphosphate; i soPPADS: Pyridoxal-5'-phosphate-6-azophenyl-2',5'-disulphonic acid; M R S 2159: Pyridoxal-5'-phosphate-6-azophenyl-4'-carboxylate; NF023: 8,8'-(carbonylbis(imino-3,1 -phenylene carbonylimino)bis(l,3,5-napththalenetrisulfonic acid); R B - 2 : reactive blue-2; T N P - A T P : 2',3'-0-(2,4,6-Trinitrophenyl) adenosine triphosphate; o x A T P : oxidized A T P ; K N - 6 2 : 1-[N,0-bis(5-Isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine;BBG:Brilliant B l u e G . 23 1.2.2.2 Subtypes of P2XR in CNS P2Xi Receptor (P2XiR): The P 2 X [ R has been found at dorsal horn spinal neurons, platelets, cerebellum and smooth muscle and is characterized by rapid desensitization. Interestingly, the P 2 X ] R is not detectable in adult rat brain, however, it has been observed in neonatal brain (Kidd et al., 1995; Collo et al., 1996). P2X2 Receptor (P2X2R): The P 2 X 2 R has been found at pre- and postsynaptic sites in the hypothalamus (Loesch et al., 1999). P 2 X 2 R show slow desensitization during several seconds of A T P application and demonstrates high sensitivity to extracellular acidity and Z n 2 + (Evans et a l , 1995). Unlike the P 2 X , R , the P 2 X 2 R does not respond to a,[3-methylene-adenosine-5'-triphosphate (a,P-meATP) (Evans et al., 1995). P2X3 Receptor (P2X3R): The P2X3R is exclusively expressed in sensory neurons of trigeminal, dorsal root and nodose ganglia, but not present in brain or peripheral tissues (Chen et al., 1995; Lewis et al., 1995). The P 2 X 3 R shows very similar characteristics to those found in the P2XiR, except that the P2X3R desensitization occurs faster than the P 2 X , R (Chen et al., 1995). P2X4 Receptor (P2X4R): The P 2 X 4 R has widespread distribution throughout the C N S and peripheral nervous system (PNS) and is highly calcium-permeable (P(Ca 2 + /Na + ) = ~4). The P 2 X 4 R shares similar properties with the P 2 X 2 R in that this receptor shows slow desensitization to ATP, no response to oc,p-meATP and sensitivity to Zn 2 + (Seguela et al., 1996). However, the concentrations of antagonists (suramin and PPADS) for the P 2 X 4 R are much higher (>100 times) than for the P 2 X 2 R to inhibit the effect of A T P (Buell et al., 1996; Wang etal., 1996). 24 P2X S Receptor (P2X5R): The P 2 X 5 R elicits approximately only 5-10% of the P 2 X i R -P2X4R-mediated currents induced by A T P (Collo et al., 1996). The angonist and antagonist profiles of P2X5R resemble those of the P 2 X 2 R (Collo et al., 1996). P2X6 Receptor.(P2X6R): The P 2 X 6 R is different from other subtypes of P 2 X R in that the latter does not form a functional homomer (Torres et al., 1999). A T P did not evoke currents in the P2X6R-transfected oocytes or human embryonic kidney 293 (HEK293) cells (Soto etal., 1996). P2X 7 Receptor (P2X 7R): A particularly unique member of the P 2 X R family is P 2 X 7 R , which can also allow permeability of large hydrophilic molecules (< 900 Da). This subtype P 2 X R generally requires higher concentrations of A T P for activation compared with the other members of the purinergic family. The P 2 X 7 R is widely expressed in macrophage-type cells such as microglia, but also mediates purinergic responses in astrocytes (Fumagalli et al., 2003) and neurons (Miras-Portugal et al., 2003). Unlike other P 2 X R subtypes, the P 2 X 7 R does not form heteromultimers (Torres et al., 1999). Rat and human P 2 X 7 R expressed in H E K 2 9 3 cells show different sensitivity to agonists. In general, human P 2 X 7 R requires higher concentrations of A T P to activate this receptor (Rassendren et al., 1997). A t higher concentrations of A T P (in excess of 1 m M ) , or using the specific P 2 X 7 R agonist, BzATP, human microglial cells exhibit an added complexity in the spectrum of inflammatory responses not evident at lower levels of ATP. 1.2.2.3 Heteromeric P2XR There have been 3 heteromeric P 2 X R identified to date including P2X2R/P2X3R, P 2 X , R / P 2 X 5 R and P 2 X 4 R / P 2 X 6 R . The P 2 X 2 R / P 2 X 3 R is activated by oc,p-meATP and 25 blocked by T N P - A T P and potentiated by acidic p H . Little or no receptor desensitization is observed. The P2X2R/P2X3R in primary afferent neurons is involved in mechanical allodynia caused by intraplantar injection of a,(3-meATP in rats (Tsuda et al., 2000). P 2 X i R / P 2 X 5 R heteromer is activated by a,(3-meATP showing a sustained current. The homomeric P 2 X ] R showed marked "run-down" when a,P-meATP is repeatedly applied at intervals of several minutes. However, no "run-down" has been observed by repeated applications of a,P-meATP to P 2 X i R / P 2 X 5 R heteromer at intervals of 10 sec. The P2X4R/P2X6R subunits are most abundantly co-expressed throughout the C N S (Collo et al., 1996). 26 1.3 PURINERGIC R E C E P T O R S IN M I C R O G L I A Molecular and immunocytochemical studies have shown the presence of P 2 Y R and P 2 X R in microglia (North, 1996, 2002; Jacobson et al., 2004). Activation of purinergic receptors in microglia triggers mobilization of intracellular [Ca 2 + ]i (reviewed in Inoue, 2002; James and Butt, 2002; Moller , 2002; McLarnon, 2005). Elevated [Ca 2 + ] , modulates a variety of intracellular signaling pathways leading to cellular functional responses such as proliferation, chemotaxis or cell death (Ferrari et al., 1997; Hide et al., 2000; Parvathenani et al., 2003). In this laboratory we have focused on microglial stimuli which lead to the release of inflammatory mediators such as pro-inflammatory cytokines. In this section, I first discuss [Ca 2 +]j mediated by P 2 Y R and P 2 X R in microglia with the latter not including P2X7R. The second section focuses on P2X7R which is a unique ionotropic purinergic receptor associated with cell apoptosis. 1.3.1 Purinergic P2YR and P2XR (not P2X7R)-mediated [Ca2+]i Mobilization A large concentration difference of C a 2 + exists between extracellular ( m M range) 94-and intracellular (nM range) regions and changes in [Ca ]i levels act as critical intracellular signaling messengers. It should first be noted that microglia show little or no 2+ + expression of voltage-gated Ca channels (Eder, 1998). However, elevated K concentration has been shown to enhance production of superoxide anion induced by phorbol 12-myristate 13-acetate in rat neonatal microglia. This result was suggested to 9+ involve voltage-gated Ca channels (Colton et al., 1994). In this laboratory the application of elevated K + to human microglia lead to no change in [Ca 2 +]j indicating the 2_j_ absence of voltage-gated Ca channels in the plasma membrane. Four major pathways 27 are reported to mediate changes in [Ca ]j levels in microglia: 1) Ca influx across the plasma membrane, 2) C a 2 + release from internal stores, 3) C a 2 + extrusion from the cytosol across the plasma membrane, 4) C a 2 + sequestration into intracellular organelles. The four 94-distinctive patterns of [Ca ], signals in cells are summarized below (Moller, 2002). 2+ (1) A transient [Ca ]j signal with a rapid increase either by release from internal stores or by influx across the plasma membrane followed by a fast decline. (2) A transient [Ca ]j signal with a rapid increase followed by prolonged plateau phase. Internal C a 2 + release is responsible for the fast increase of [Ca 2 +]j and the secondary [Ca ]j signal is attributable to influx via receptor-operated channels (ROC) or store-operated channels (SOC). The latter mechanism is a characteristic feature of non-excitable cells (Putney, 1986; Hoth and Penner, 1992). (3) Oscillatory [Ca 2 +]j signals. This type of [Ca 2 + ]i signal is generated by a complex interplay between [Ca 2 + ] i from internal stores and [Ca 2 + ] ; influx via S O C . 2+ * * (4) Slowly increasing [Ca ]j signals. Activation of a low plasma membrane conductance or perturbed C a 2 + extrusion mechanism are involved in this type of [Ca 2 + ] i signal. 1.3.2 Store-operated Channels (SOC) in Microglia S O C are Ca permeable channels in the plasma membrane and are highly expressed in microglia. SOC-mediated Ca entry, with dependence on the amount of Ca depletion from internal stores, was first reported in parotid acinar cells (Putney, 1986). This process was originally called capacitative Ca 2 + entry. Thapsigargin, a specific inhibitor of sarcoplasmic/endoplasmic reticulum Ca 2 + -ATPase ( S E R C A ) , was used to deplete internal stores. Following store depletion some unknown signal caused activation 28 of SOC-mediated Ca entry through plasma membrane. A number of models have been developed to link store depletion with activation of S O C (reviewed in Parekh and Putney, 2005). A t present, however, the specific signals linking store depletion with S O C -mediated C a 2 + entry, in microglia are not well understood. I found acute application of A T P to human microglia (at 100 uM) caused a rapid, transient increase in [Ca 2 +]j (Figure 1-2A; see legend for use of F340/F380 ratio as an 2_|_ indication of [Ca ]j). The response showed the decay phase of the trace to be well-fitted by a single exponential. A similar response (Figure 1-2B) was also evoked by A T P application in the absence of external C a 2 + (Ca 2 +-free PSS), however, the duration was shorter compared to the A T P response in medium containing C a 2 + . These results suggest that depletion of C a 2 + from E R stores, secondary to A T P binding to P 2 Y R , accounts for the majority of increased [Ca 2 + ] ; (see model Figure 1-4). The small difference in time course between A T P responses in Figure 1-2A (PSS) and Figure 1-2B (Ca 2 +-free PSS) could be due to activation of some influx pathway. In separate experiments, I confirmed that this pathway represented a low level of C a 2 + influx through S O C . A s shown in the representative trace in Figure 1-2C, pretreatment with the S O C inhibitor SKF96365 acted to shorten the duration of the ATP-induced response to that found in control (Choi et al., 2003). Subsequent experiments were designed to investigate possible interactions between P 2 X R and P 2 Y R . I used the compound pyridoxal-phosphate-6-azophenyl-2\ 4"-disulfonic acid (PPADS) as a selective P 2 X R antagonist. P P A D S is reported as a specific inhibitor of P 2 X R when applied to microglia at concentrations below 10 u M (Choi et al., 2003). The interesting result was that the duration of an A T P response with P P A D S 29 pretreatment was markedly prolonged compared to the control A T P response (see Figure 4-1 A , B) . I then found that the prolonged phase of the A T P response with P P A D S was abolished in the absence of extracellular C a 2 + or using SKF96365 to block S O C (see Figure 4-2A, B) . 30 1.0 i 0.8 o oo 0.6 o 0.4 0.2 0.0 -B 1.0 0.8 o 0 0 £ 0.6 § 0.4 0.2 0.0 A T P (PSS) 100 200 300 Time (sec) A T P (Ca 2 +-free PSS) 100 200 300 Time (sec) 1.0 o0.8 0 0 tjn 0.6 | 0.4 0.2 0.0 SKF96365 A T P (PSS) 100 200 Time (sec) 300 Figure 1-2. A T P (applied at 100 uM)-induced changes in [Ca 2 +] f in human microglia A: A T P applied in Ca 2 +-containing PSS. B: A T P applied in Ca 2 +-free PSS. C: A T P applied in Ca 2 +-containing PSS in the presence of SKF96365 (50 uM) . See section 2.2.5 for use of F340/F380 ratio as a measure for [ C a 2 + ] . 31 The results indicate that in standard solution, A T P (applied at 100 uM) causes a 2+ P2YR-dependent release of Ca from E R stores with little SOC. However, block of P 2 X R with P P A D S leads to a substantial SOC influx of C a 2 + . The results were interpreted as indicating that in control, A T P binding to some P 2 X R attenuates a secondary phase of [Ca 2 + ] i due to entry of C a 2 + through SOC. The reasoning was that under control conditions A T P binding to some P 2 X R (but not P 2 X 7 R ) mediated cell depolarization due to influx of N a + and this depolarization acted to inhibit entry of C a 2 + through S O C . In the presence of P P A D S block of P 2 X R , cell depolarization was inhibited and S O C entry o f Ca was established. In essence, the A T P response prolonged by P P A D S was due to a S O C component. This possibility was confirmed using adenosine diphosphate-P-S ( A D P -P-S), a selective agonist for P 2 Y R . Application of ADP-P-S, in the absence of any contribution from activation of P 2 X R , caused prolongation of response compared with control A T P responses (see Figure 4-1C). The ADP-P-S-induced prolongation of response was blocked in the presence of SKF96365 (data not shown). Overall, the duration of ADP-P-S responses was increased almost two-fold compared with time courses of A T P responses (Choi et al., 2003). This work is detailed in chapter 4. Independent experiments have documented that cell depolarization, subsequent to elevation of external K + , also inhibits SOC. Elevated external K + reduced S O C influx in accordance with changes in electrochemical driving force over a limited concentration range of the cation (McLarnon et al., 2000). However, at high concentrations of K + (above 35 mM) , SOC entry of Ca was abolished at potentials that would still yield a large inward driving force. One possible reason for this result is that depolarization-induced inactivation of the capacitative C a 2 + entry channel occurs at larger cell 32 depolarizations. Interestingly, evidence for a depolarization-induced gating effect to inactivate SOC has been reported in endothelial cells (Wang and Van Breemen, 1999). The acidification of external solution is also reported to reduce S O C activity in human microglia (Khoo et al., 2001). This work suggested that acidic p H modified cell potential possibly by actions on pH-sensitive chloride (CI") channels. Since maxi-conductance type CI" channels were reported in human microglia (McLarnon et al., 1997), the results could suggest acidification effects on these CI" channels as a source of cell depolarization (Khoo et al., 2001). The novel findings described above suggest that interactions between P 2 X R and P 2 Y R pathways modulates purinergic (ATP at < 100 uM)-induced changes in [Ca 2 +]j . A s noted above, a plausible explanation for these effects is that A T P binding to a P 2 X R other than P 2 X 7 R leads to cell depolarization and block of SOC-mediated C a 2 + entry. The latter is triggered by E R depletion resulting from A T P binding to P 2 Y R . In this case influx of N a , rather than Ca , predominates in the activated P 2 X R . Thus, the P 2 X R underlying this effect would not likely be a P 2 X 7 R subtype since this purinergic receptor allows a high influx of C a 2 + . In addition, P 2 X 7 R is only activated by high levels of A T P (> 1 mM)(see following section). R T - P C R analysis has been used to examine expression of subtype P 2 X R in human microglia. A representative R T - P C R is shown in Figure 1-3. The results show a strong band for constitutive expression of P 2 X 4 R in human microglia. N o evidence for expression of any other P 2 X R was found in the R T - P C R study (data not shown). Thus, P 2 X 4 R is suggested as a candidate for the P 2 X R which mediates cell depolarization, thereby inhibiting a SOC component in. A T P stimulation (at 100 uM) of microglia. 33 G A P D H Figure 1-3. Expression of subtype of P2XR in human microglia P 2 X 4 R and P 2 X ? R are constitutively expressed in human microglia. N o bands were identified for other P 2 X R (data not shown). Fetal brain tissues are used as positive control. G A P D H is a reaction standard. 34 A schematic model has been developed from the experimental results described above. This model includes separate pathways dependent on the levels of A T P used to stimulate human microglia. Details of the model are presented in Figure 1-4. 35 Figure 1-4. Simplified schematic diagram of purinergic-mediated C a 2 + mobilization in human microglia A t lower levels 100 MM), A T P acts via P 2 Y R to increase [ C a 2 ^ by release from endoplasmic reticulum (ER) stores; a signal associated with depletion of E R triggers influx of C a 2 + mediated by store-operated channels (SOC). A T P also acts v i a some ionotropic family member, P 2 X ( 1 6 ) R , to mainly pass inward N a + current leading to cellular depolarization and inhibition of SOC-mediated entry o f C a 2 + . Influx of C a 2 + through S O C is also enhanced or diminished by changes in membrane potential dependent on the patterns of anion or K + currents (not shown). At higher concentrations of A T P (in excess of 1 mM) , activation of P 2 X 7 R in human microglia can lead to a marked enhancement in [Ca 2 + ] ; . Membrane transporters, such as N a + / C a 2 + exchanger, are not included in the diagram. Solid line: activating signal; Dashed line: inhibiting signal. 36 1.3.3 P2X7R-mediated Signal Transduction Pathways This section focuses on microglial P2X7R which is a unique ionotropic purinergic receptor. A s shown in the model (Figure 1-4), P2X7R in human microglia is activated with higher concentrations of A T P (in excess of 1 mM) . It should be noted that macrophage-type cells including resident microglia are the primary cell types expressing P 2 X 7 R in the C N S . Activation of P2X7R in microglia stimulates a host of intracellular signaling pathways and modulates cellular responses such as production of inflammatory and neurotoxic compounds. P2X 7R-mediated intracellular signal transduction pathways in microglia are summarized below. A T P (1 m M ) mediated production of T N F - a and associated intracellular signaling pathways have been studied in rat microglia (Hide et al., 2000). This study revealed that ATP-mediated expression and production of T N F - a in cultured rat microglia was [Ca 2 +]j dependent. The selective P 2 X 7 R agonist, B z A T P also caused release of T N F - a in cultured rat microglia. Signaling transduction pathways in ATP-induced T N F - a release included E R K - 1 / 2 and p 3 8 M A P K . Inhibitors of E R K - 1 / 2 and p 3 8 M A P K , PD98059 and SB203580 respectively, effectively attenuated ATP-induced T N F - a synthesis. Ferrari et al (1997) reported that the bacterial endotoxin, L P S , induces microglial secretion of A T P , which in turn activates P 2 X 7 R leading to production of the mature form of I L - i p i n microglial cell lines, N 9 and N13. O x A T P , a P 2 X 7 R antagonist, prevented LPS-induced secretion of I L - i p by inhibiting depletion of endogenous K + in macrophages and microglia. I L - i p converting enzyme (ICE), a cysteine protease, plays an important role in cleaving pro-IL- ip into mature form of I L - i p . Depletion of cytoplasmic K + increases ICE activity resulting in maturation of I L - i p . 37 Purinergic receptor mediated production of superoxide (0~2) has been studied using rat primary microglia (Parvathenani et al., 2003). The results showed that A T P and B z A T P stimulate 0~2 production through P2X7R. Activation of N A D P H oxidase was involved in production of O 2 and both E R K - 1 / 2 and p 3 8 M A P K were activated in response to B z A T P . However, only inhibition of p 3 8 M A P K using SB203580 diminished 0 _ 2 production. Superoxide production may be dependent on phosphatidylinositol 3-kinase (PI3-K). Pharmacological inhibition of PI3-K with a selective inhibitor, LY294002, was found to significantly reduce 0~2 production (Parvathenani et al., 2003). Activation of P 2 X 7 R is associated with large pore formation increasing cell permeability and subsequently leading to microglial activation. Two distinct hypotheses are used to explain P2X 7 R-mediated pore formation: dilation hypothesis and separate pore hypothesis (Liang and Schwiebert, 2005). The former hypothesizes conformational changes of P 2 X 7 R upon activation and the latter suggests that P2X 7 R-mediated intracellular signaling molecules^such as calcium or M A P K s activate a separate channel entity. P2X 7 R-mediated pore forming activity was attenuated in reduced extracellular calcium and in the absence of [Ca 2 + ] , by using chelating agents (Faria et al., 2005). Recent studies provided evidence that p38 M A P K and Rho activation, but not E R K - 1 / 2 , is involved in P2X 7 R-mediated actin rearrangement and membrane blebbing in R A W 264.7 macrophages (Pfeiffer et al., 2004). Inhibition of Rho effector kinase using Y-27632 completely prevented P2X 7R-dependent blebbing in HEK293 cells transfected with P 2 X 7 R . Hexokinase, an ecto-ATPase did not induce blebbing in H E K - P 2 X 7 R cells suggesting little or no contributions from A T P degradation products such as glucose-6-38 phosphate, A D P , A M P and adenosine. O x A T P or removal of extracellular calcium inhibited Webbing in H E K - P 2 X 7 R cells. 2-arachidonoylglycerol (2-AG) is one of the most abundant endogenous cannabinoid ligands (endocannabinoids) and plays a critical role in neuroinflammation. The activation of P 2 X 7 R either using a high concentration of A T P or B z A T P markedly increased the production of 2 - A G (Witting et al., 2004). Sustained increase of [Ca 2 + ] i by activating P 2 X 7 R leads to increased activity of diacylglycerol lipase ( D G L ) which systematically increases production of 2 - A G and inhibits activity of monoacylglycerol lipase ( M G L ) . The latter is an enzyme that degrades 2 - A G . It has been also reported P2X 7 R-mediated 2 - A G production occurred to a lesser extent (about 5% of microglial 2-A G production) in rat primary astrocytes (Walter et al., 2004). However, it was noted that contribution of astrocytes to 2 - A G production in brain parenchyma should not be disregarded in neuropathological conditions because astrocytes are the most abundant cell types in the brain parenchyma. Activated microglia can also provide neuroprotection by releasing trophic factors such as plasminogen (Inoue et al., 1998). This neurotrophic factor is known to promote the development of mesencephalic dopaminergic neurons and neurite outgrowth from explants of neo-cortical tissue. Inoue et al (1998) have reported Ca -dependent production of plasminogen from ATP-stimulated microglia with o x A T P pretreatment inhibiting [Ca 2 + ]i increase and levels of plasminogen. These results link activation of P 2 X 7 R and increased [Ca ]i with microglial release of plasminogen which may confer neuronal protection. 39 1.4 R E S E A R C H HYPOTHESIS Microgl ia are resident brain cells mediating chronic inflammatory responses in brain leading to progressive neuronal damage in neurodegenerative diseases. A T P serves an important role as a signaling factor for microglial activation and the subsequent induction of cellular responses in inflamed brain. The hypothesis of the proposed research is that activation of P 2 X 7 R in microglia is a critical factor in mediating chronic brain inflammation and modulation of P 2 X 7 R , including downstream C a 2 + signaling pathways, alters the expression and production of pro-inflammatory mediators in stimulated human microglia. Pharmacological modulation of P 2 X 7 R and other purinergic receptors has been investigated as a potential therapeutic intervention in decreasing inflammatory responses in microglia in vitro and in vivo. 40 1.5 S U M M A R Y O F PROPOSED R E S E A R C H O B J E C T I V E S The overall thesis objective is to investigate roles of P 2 X 7 R in mediating brain inflammation and purinergic receptor-dependent intracellular C a 2 + signaling pathways and functional responses in microglia both in vitro and in vivo. The specific objectives are listed below: 1. To investigate roles of P 2 X 7 R in an in vivo animal model of inflammation using injection of L P S into rat striatum and to determine effects of L P S stimulation of cultured human microglia in vitro on cellular expression of inflammatory mediators and properties of Ca2 +-dependent signaling pathways. To determine effects of the P 2 X 7 R antagonist o x A T P as a modulator of inflammation in vivo and in vitro. 2. To examine the expression pattern of P 2 X 7 R in a broad spectrum of inflammatory conditions related to A D . These include P 2 X 7 R expression in adult microglia from Alzheimer's disease (AD) patients and non-demented (ND) individuals, P 2 X 7 R expression and altered functional responses in A ( 3 M 2 peptide-stimulated human fetal microglia and in vivo properties of P 2 X 7 R with intrahippocampal injection of A P 1 . 4 2 in an animal model of A D . 3. To investigate the effects of inhibition of P 2 X R other than P 2 X 7 R on C a 2 + signaling pathways and expression and production of cyclooxygenase-2 (COX-2) in human microglia. 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Biochem Pharmacol 59(2):203-9. 53 C H A P T E R 2: M O D U L A T I O N O F T H E PURINERGIC P2X 7 R E C E P T O R A T T E N U A T E S L P S - M E D I A T E D M I C R O G L I A L A C T I V A T I O N A N D N E U R O N A L D A M A G E 1 2.1 INTRODUCTION Inflammation is a critical component in the progressive neurodegeneration manifest in neurological disorders. The activation of glial cells, microglia and astrocytes is a characteristic finding in brain inflammation. Microglia, as the immunocompetent resident cells of the brain, possess properties particularly suitable in mediating cellular inflammatory responses. The release of pro-inflammatory mediators from activated microglia has been suggested as a contributing factor to pathology in disease (McGeer and McGeer, 1998; Lue et al., 2001; Walker et al., 2001) with anti-inflammatory agents conferring some degree of neuroprotection (Suzuki et a l , 2004; Klegeris et al., 2005). A n important aspect in the induction of an inflammatory response may be a signal from damaged neurons to activate glia. Pathological conditions associated with spinal cord injury and ischemia are characterized by high extracellular levels of A T P (Le Feuvre et al., 2002; Franke et al. 2004; Wang et al., 2004). Microgl ia (and astrocytes) express purinergic receptors for the two receptor subfamilies, ionotropic (P2XR) and metabotropic (P2YR), which mediate mobilization of intracellular C a 2 + [Ca 2 +]j (Moller, 2002; McLarnon, 2005). A particularly interesting member of the P 2 X R family is P 2 X 7 R , principally expressed by macrophage-like cells of the brain, and linked with a high conductance pore. Activation of P 2 X 7 R generally requires elevated levels of extracellular A T P (in excess of 1 m M ) suggesting an involvement in pathophysiological conditions. Upon activation, P 2 X 7 R mediates influx 1 A version of this chapter has been submitted for publication. Choi HB, Ryu JK, Kim SU, McLarnon JG. Modulation of the Purinergic P2X 7 Receptor Attenuates LPS-mediated Microglial Activation and Neuronal Damage. 54 of N a and Ca and concomitant efflux of K which in microglia helps shape and modulate a diversity of cellular inflammatory responses (Gudipaty et al., 2003). Functionally, P2X7R plays a critical role in microglial release o f the pro-inflammatory cytokine IL-1(3 by reducing intracellular levels of K + (Ferrari et al., 1999; Rothwell and Luheshi, 2000; Humphreys et al., 2000). In addition, P 2 X 7 R is coupled to multiple caspases and stress activated protein kinases ( S A P K ) leading to cellular apoptosis (Ferrari et al., 1999; Humphreys et al., 2000). Recent work has reported activation of P 2 X 7 R in microglia increased production of superoxide anion ( O 2 ) in a transgenic Tg2576 mouse model of Alzheimer's disease (Parvathenani et al., 2003). Effects of P 2 X 7 R as a mediator of brain inflammatory responses have also been documented. The specific P 2 X 7 R ligand, B z A T P , increased production of pro-inflammatory cytokines in stimulated macrophages and microglial cells (Rampe et al., 2004) and increase interferon-y (IFN- y)-induced phosphorylation of extracellular signal-regulated kinases-1/2 (ERK-1/2) in murine B V - 2 microglial cells (Gendron et al., 2003). The inhibition of P 2 X 7 R using oxidized A T P (oxATP) or pyridoxal-phosphate-6-azophenyl-2', 4"-disulfonic acid (PPADS) effectively improved recovery after spinal cord injury (Wang et al., 2004). In this study we have investigated the roles of P 2 X 7 R , in the absence and presence of o x A T P , in an LPS-animal m o d e l o f inflammation and in vitro in LPS-stimulated human microglia. In vivo, a spectrum of oxidative stress markers have been measured including inducible nitric oxide synthase ( iNOS), 3-nitrotyrosine (3-NT, indicative of peroxinitrite-mediated nitration), 4-hydroxynonenal (4-HNE, indicative of lipid peroxidation) and 8-hydroxy-2'-deoxyguanosine (8-OHdG, indicative of oxidative D N A c . 55 damage). In vitro analysis has included study of P 2 X 7 R involvement in expression of inflammatory mediators and changes in [Ca 2 +]j mobilization in L P S activated microglia. 56 2.2 M A T E R I A L S AND M E T H O D S 2.2.1 Animals A l l animal experiments were carried out in accordance with the guidelines set by the Committee on Animal Research at the University of British Columbia. Male, 6-week-old, Sprague-Dawley rats (240-260 g) were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and then mounted in a stereotaxic apparatus (Kopf Instruments, Tujunge, C A ) . Animals received unilateral stereotaxic injection of 5 L P S (dissolved in PBS) over 4 min at the following coordinates; A P : +1.0 mm, M L : -2.6 mm, D V : -5.0 mm (Paxinos and Watson, 1998). O x A T P (Sigma, St. Louis, M O ) was dissolved in water and further diluted to P B S and animals received intracerebroventricular (icv) injection 30 min prior to L P S injection. Control animals received striatal injection of P B S . The animals were deeply anesthetized with sodium pentobarbital at 6 hr, 12 hr, 24 hr, or 3 day post-L P S injection and transcardially perfused with heparinized saline followed by 4% paraformaldehyde for further analysis. 2.2.2 Cell Culture The methods used in the isolation and identification of microglia have been described (Satoh and K i m , 1995; Nagai et al., 2001). In brief, human embryonic brain tissues (12-18 weeks of gestation) were used for the preparation of microglia. Brain tissues were dissected into small blocks, incubated in phosphate buffered saline (PBS) containing 0.25% trypsin and 40 |j,g/ml DNase for 30 min at 37°C and dissociated into single cells by repeated pipetting. Dissociated cells were plated in T75 flasks in a medium consisting of Dulbecco's modified Eagle's medium ( D M E M ) with high glucose 57 containing 5% horse serum, 25 p.g/ml gentamicin, and 2.5 p.g/ml amphotericin B . Freely floating microglia were harvested from a medium of mixed cell cultures after 7-10 days of growth in culture flasks and plated on aclar coverslips for identification, on poly-L-lysine-coated glass coverslips for calcium spectrofluorometry or plated on six-well multiplates for R T - P C R . Immunostaining was performed on isolated cells using C D l i b and ricinus communis agglutinin ( R C A ) , specific markers for microglia, and the purity of human microglia was in excess of 98% (Nagai et al., 2001). The use of embryonic human tissues was approved by the Clinical Screening Committee for Human Subjects of the University of British Columbia. 2.2.3 Immunohistochemistry Free-floating sections (40-um serial coronal sections) were processed for immunostaining as previously described (Ryu et al., 2003) with the following primary antibodies; N e u N for neurons (1: 500; Chemicon, Temecula, C A ) , OX-42 for microglia (1: 400; Serotec, Oxford, U K ) , G F A P for astrocytes (1: 500; Sigma), P 2 X 7 R (1:200; Alomone, Jerusalem, Israel), 3-nitrotyrosine (3-NT) (1: 500; Upstate, Lake Placid, N Y ) , 4-hydroxynonenal (4 -HNE) ( l : 500; Jaica, Shizuoka, Japan), 8-hydroxy-2'-dedxyguanosine (8-OHdG) (1: 500; Jaica), iNOS (1: 100; Upstate) and cleaved caspase-3 (1:200; Cel l Signaling Technology, Beverly, M A ) . Phospho-p38 M A P K (1: 250; Cel l Signaling Technology) and p65 (1: 250; Santa Cruz, C A ) were used for intracellular signaling markers. Sections were incubated at room temperature (RT) for 2 hr in biotinylated anti-IgG (1: 200; Vector, Burlingame, C A ) followed by an A B C elite system (1: 200; Vector) for 1 hr. The reaction products were developed using a D A B 58 (diamine-benzidine) kit (Sigma). Alexa Fluor 594 anti-rabbit and Alexa Fluor 488 anti-mouse IgG (1:100) secondary antibodies (Molecular Probes, Eugene, OR) were used for immunofluorescent staining. Appropriate positive and negative control experiments were done; in each immunohistochemical staining the primary antibody was omitted to assess for nonspecific binding of the secondary antibody. For 3-NT immunohistochemistry, positive (use of 24 m M peroxynitrite) and negative (use of 10 m M nitrotyrosine) controls were performed (Ayata et al., 1997). The specificity for P 2 X 7 R antibody may not be unique since immunoblotting results have shown a trace of P 2 X 7 R expression in P 2 X 7 R -deficient mice (Auger et al., 2005). 2.2.4 R T - P C R For in vivo experiments animals were deeply anesthetized and sacrificed by i decapitation at different time points (0, 1, 4, 8, 12, 24, 48 hrs) after L P S injection. A s a control, brains were used at 12 hr post-injection of P B S . Brains were removed and the striatum was dissected onto a cold metal tissue matrice (Harvard^ Apparatus, Montreal, Quebec, Canada). The tissue samples were frozen in liquid nitrogen. Total R N A was then extracted from frozen striatal tissue using TRIzol reagent (Gibco-BRL, Gaithersburg, M D ) subjected to DNase I treatment and complementary D N A ( c D N A ) synthesis was carried out using M - M L V reverse transcriptase (Gibco-BRL) . M - M L V reverse transcriptase was omitted as a negative control. For in vitro experiments, human microglia were plated on 12-well plates and were incubated in serum-free medium for 48 hr. This procedure yielded a homogeneous population of cells with ramified, process-bearing morphology. Cells were then treated with L P S (100 ng/ml for 8 hr) in the absence 59 and presence of o x A T P (300 u M applied 2 hr prior to L P S application). P C R primers are all intron-spanning and sequences and expected product sizes are listed in Table 2-1. P C R conditions were as follows: initial denaturation at 95°C for 6 min followed by 35 cycles of denaturation at 95°C for 45 s, annealing at 55-60°C for 1 min and extension at 72°C for 1 min. A final extension was carried out at 72°C for 10 min. Glyceraldehyde-3-phosphate dehydrogenase ( G A P D H ) and p-actin were used as reaction standards for human and rat samples, respectively. For semi-quantifications of P C R products, cycle numbers were chosen during the exponential phase of the reaction. The amplified D N A s were identified using 1.5% agarose gels containing ethidium bromide (EtBr) and visualized under U V light. The band intensities of P C R products in control and with stimuli were measured using N I H Image J 1.24 software (National Institutes of Health, Bethesda, M D ) and expressed as relative m R N A levels ( m R N A values normalized to G A P D H or P-actin). 60 ( Table 2-1. Gene specific P C R primer sequences Gene Sequence (5 -^3') Product Size (bp) C O X - 2 sense T T C A A A T G A G A T T G T G G G A A A A T T G C T 305 C O X - 2 antisense A G A T C A T C T C T G C C T G A G T A T C T T G A P D H sense C C A T G T T C G T C A T G G G T G T G A A C C A 251 G A P D H antisense G C C A G T A G A G G C A G G G A T G A T G T T C I L - i p sense A A A A G C T T G G T G A T G T C T G G 179 I L - l p antisense T T T C A A C A C G C A G G A C A G G IL-6 sense G T G T G A A A G C A G C A A A G A G G C 159 IL-6 antisense C T G G A G G T A C T C T A G G T A T A C IL-8 sense A T G A C T T C C A A G C T G G C C G T G 301 IL-8 antisense T A T G A A T T C T C A G C C C T C T T C A A A A IL-10 sense G A T C T C C G A G A T G C C T T C A G C A G A 194 IL-10 antisense C C T T G A T G T C T G G G T C T T G G T T C T IL-12 sense T C A C A A A G G A G G C G A G G T T C T A A G C 213 IL-12 antisense C C T C T G C T G C T T T T G A C A C T G A A T G M C P - 1 sense A C T G A A G C T C G C A C T C T C 348 M C P - 1 antisense C T T G G G T T G T G G A G T G A G P 2 X 7 R sense A C A A T G T T G A G A A A C G G A C T C T G A 728 P 2 X 7 R antisense C C G G C T G T T G G T G G A A T C C A C A T C T G F - p 1 sense T T G C A G T G T G T T A T C C G T G C T G T C 185 T G F - p 1 antisense C A G A A A T A C A G C A A C A A T T C C T G G T N F - a sense C A A A G T A G A C C T G C C C A G A C 490 T N F - a antisense G A C C T C T C T C T A A T C A G C C C P-actin(r) sense G T G G G G C G C C C C A G G C A C C A 526 P-actin(r) antisense G T C C T T A A T G T C A C G C A C G A T T T C iNOS.(r) sense C C T G C C C C T T C A A T G G T 758 i N O S (r) antisense G G T A T G C C C G A G T T C T T T P 2 X 7 R (r) sense A G G A G C C C C T T A T C A G C T C T 692 P 2 X 7 R (r) anti sense C A T T G G T G T A C T T G T C G T C C (r) represents rat specific primers and other primers are human specific sequences. 61 2.2.5 Ca Spectrofluorometry The procedures used in Ca 2 +-sensitive fluorescence microscopy in this laboratory have been previously described (Khoo et al., 2001; McLarnon et al., 2001). In brief, the fluorescent C a indicator fura-2 acetoxymethylester (Fura-2/AM) was added with pluronic acid (both agents at 1 uM) to cultured human microglia bathed in physiological saline solution (PSS) at R T (20-22°C) for 30 min. Cells were washed for 10 min in dye-free PSS. Coverslips were placed on the stage of an inverted microscope (Zeiss Axiovert with *40 quartz objective) and alternating 340 and 380nm U V light was used for excitation. Fluorescent signals were recorded by passing 510nm of emission light (bandwidth of 40nm) to a digital C C D camera (Retiga 13001, Burnaby, Canada). A n imaging system (Empix, Mississauga, O N , Canada) was used to process and analyze fluorescent signals based on the wavelength ratios (340/380nm) and ratio images were processed every 6 s. Values for ratios are reported with no conversion to absolute levels of C a 2 + . In M n 2 + quenching studies, the divalent cation M n 2 + (at 25 uM) was added to the Ca -free PSS. In the quenching experiments an excitation wavelength of 360nm was used since this wavelength corresponds closely to the isosbestic point of the fura-2/AM spectrum. Physiological saline solution (PSS) contains (in m M ) the following: N a C l (126), KC1 (5), C a C l 2 (1), M g C l 2 (1.2), H E P E S (10), D-glucose (10); p H 7.4. Ca 2 +-free PSS (0PSS) contains the same ionic composition as PSS with the exception that it included E G T A (at 1 m M ) with no added CaCL;. A l l imaging experiments were done at RT . 62 2.2.6 Quantitative Analysis The four immunostained sections (AP: +1.4, +1.2, +1.0, and +0.8) were digitized and analyzed using image analysis program N I H version 1.57 (Wayne Rasband, NIH) as described previously (Ryu et al., 2004). Quantification of neuronal damage in the striatum was performed by counting neurons double stained with N e u N and caspase-3 using a Zeiss Axioplan 2 fluorescent microscope ( x 40 objective). The number of N e u N (+)ve and caspase-3 (+)ve cells was counted and expressed as caspase-3 immunoreactive neurons/mm 2. The number of N e u N (+)ve cells were counted for treated and control groups to determine neuronal loss. OX-42 immunoreactive cells were counted for the measurement of microglial activation (Robinson et al. 1986). For the measurement of oxidative stress, areas containing 4 - H N E or 8-OHdG-immunostained cells were sharply delineated, measured in four sections of the striatum, and expressed as 4 - H N E - or 8-OHdG-immunoreactive areas. For the quantification of iNOS and 3-NT, immunoreactive cells were counted from four sections of the striatum. Quantitative analyses were done in a blinded manner. 2.2.7 Statistics and Analysis A l l results are expressed as means ± S . E . M . Statistical significance of differences for among group comparisons was evaluated using one-way analysis of variance. ( A N O V A ) followed by Newman-Keuls post hoc multiple comparison test (GraphPad Prism 3.0). Significance levels were set at P < 0.05 for all tests. 63 2.3 R E S U L T S 2.3.1 Effects of LPS on P2X 7 R Expression and Production in Rat Brain Activation of the purinergic receptor subtype P 2 X 7 R contributes to inflammatory responses in the C N S (Rathbone et al., 1999). L P S is a potent stimulus to induce inflammation and we initially examined the effects of L P S on P 2 X 7 R expression in rat brain using R T - P C R and immunohistochemical analysis. L P S (5 jag) was injected into rat striatum and expression of P 2 X 7 R was determined at different post-injection time points (1, 4, 8, 12, 24, 48 hrs). Representative R T - P C R results (Figure 2-1 A ) showed no evident P 2 X 7 R in control (12 hr P B S injection). However, expression of P 2 X 7 R increased in a time-dependent manner to a maximum level at 12 hr post-injection of L P S . Little or no expression of the purinergic receptor was found at the longest experimental time of 48 hr after L P S injection. Previous work has reported upregulated microglial P 2 X 7 R after 1 and 4 d post-i shemic insult using immunohistochemistry (Franke et al., 2004). We have chosen a time 24 hr L P S post-injection for immunohistochemical analysis using ant i -P2X 7 R antibody since production of P 2 X 7 R was most significantly upregulated at this time point. In control, P B S (24 hr) was injected and induced little or no P 2 X 7 R expression (left panel, Figure 2-1B). However, L P S injection (right panel, Figure 2-1B, green staining) upregulated expression of P 2 X 7 R compared to PBS-injected brain. Overall, results are presented in the bar graph of Figure 2-1C. Immunoreactivity (ir) to P 2 X 7 R for PBS and LPS-injected groups was 1.0 ± 0.5/mm 2 and 36.0 ± 3.8/mm 2, respectively (n=4 animals): These results show marked upregulation of expression and production of P 2 X 7 R in the presence of the inflammatory stimulus, L P S . 64 Figure 2-1. LPS induces expression and production of P2X7R in rat striatum A : L P S injection (5 ixg) into rat striatum induced time-dependent expression of P 2 X ? R m R N A showing maximum expression at 12 hr post-injection. B: Brain sections were immunostained with P 2 X ? R antibody 24 hr post-injection with P B S (left panel) or L P S (right panel). P 2 X ? R (+)ve cells are shown in green. Scale bar, 25 um. C : The bar graph shows the number of P 2 X ? R ir cells post- P B S and L P S injection (for 24 hr) (n=4 animals). *P < 0.05 compared to PBS-injected striatum. 65 2.3.2 Co-localization of P2X 7 R with Glial Cells in Rat Brain P 2 X 7 R is highly expressed in glial cells under inflammatory conditions (Collo et al., 1997) with only a few reports of the presence of P 2 X 7 R in neurons (Deuchars et al., 2001; Franke et al., 2004). Cellular expression of P 2 X 7 R was examined at 24 hr post-injection of L P S (or P B S as control) using double immunostaining for cell-specific markers (NeuN for neurons, G F A P for astrocytes, OX-42 for microglia; red cellular staining) with P 2 X 7 R (green staining). A representative staining pattern is presented in Figure 2-2A showing L P S induced a predominant co-localization of P 2 X 7 R with microglia and to a lesser extent with astrocytes; no evident immunostaining for P 2 X 7 R was found in neurons. Quantification of cellular P 2 X 7 R ir is shown in Figure 2-2B (n=4 animals). With L P S injection, the maximum P 2 X 7 R ir was found in microglia (42.7% of cells showing expression of P 2 X 7 R ) with a lesser ir measured in astrocytes (22.5% of cells with P 2 X 7 R expression); the corresponding values in PBS-injected animals were 10.3% (microglia) and 8.5%o (astrocytes). Neurons showed minimal ir for P 2 X 7 R with respective values of 1%> (LPS injection) and 0.7% (PBS injection). Thus, expression of P 2 X 7 R is primarily upregulated in microglia and to a lesser extent in astrocytes in LPS-injected rat striatum. 66 * G F A P OX-42 Figure 2-2. P2X ? R is co-localized with microglia and astrocytes in rat brain A : Brain tissues were double immunostained using cell-selective markers N e u N (for neurons, left panel), G F A P (for astrocytes, middle panel) and OX-42 (for microglia, right panel) 24 hr post-LPS injection into rat striatum. Cel l specific markers are presented in red and P 2 X 7 R is shown in green. Scale bar, 20 urn. B: The bar graph shows % o f P 2 X ? R (+)ve cells co-localized with cell selective markers NeuN, G F A P and OX-42 after P B S or L P S injection into rat striatum (n=4 animals). *P < 0.05 compared to P B S -injected striatum. B 60 50 5 40 r-x 30 2 20 10 0 • PBS • LPS NeuN 67 2.3.3 Effects of oxATP on LPS-mediated Microglial Activation LPS-induced activation of microglia is associated with a change in morphology from mainly ramified (characterized by a small cell body and fine processes) to ameboid (swollen cell body and retracted processes) shape and by marked enhancement in numbers of microglia (Iravani et al., 2005). We have, examined morphological changes and also the number of OX-42 (+)ve microglia in the presence of o x A T P , an inhibitor of P 2 X 7 R , injected intracerebroventricularly (at 1 u M ) 30 min prior to L P S injection. Representative immunostaining for OX-42 is presented in Figure 2-3 (A-C) and shows P B S injection is mainly associated with a ramified morphology indicative of resting cells (Figure 2-3A). LPS-injection (at 5 ug for 3 d) induced an evident shift to an ameboid morphology with cells displaying roundish cell bodies and shortened processes (Figure 2-3B) suggestive of an activated state. Additionally, numbers of OX-42 (+)ve cells were increased in LPS-injected striatum (Figure 2-3B). O x A T P treatment (at 1 u.M) was effective in diminishing the levels of OX-42 (+)ve microglia in LPS-injected striatum (Figure 2-3C). OX-42 immunoreactive cells per m m 2 were counted for each treatment with overall results presented in Figure 2-3D. The numbers of microglia were almost tripled with L P S injection relative to PBS-injected striatum (140.7 + 4.1/mm 2 and 51.2 ± 2.0/mm , respectively). In the presence of o x A T P (at 1 uM) with L P S the corresponding numbers of OX-42 (+)ve cells were 92.3 + 3.3/mm 2 (n=5 animals) representing a significant decrease (by 34.4%) compared to L P S alone. O x A T P alone did not induce morphological changes (data not shown). These results suggest involvement of P 2 X 7 R in mediating LPS-induced microglial proliferation. 68 o ' t t' • * ' '' * P B S L P S L P S + o x A T P D 160 120 u 80 P B S LPS L P S + o x A T P Figure 2-3. OxATP reduces the number of activated microglia in LPS (5 [ig for 3 d) injected rat brain Brain sections were immunostained with OX-42 antibody post-injection with P B S (A), L P S (B) or LPS with o x A T P (C). O x A T P (at l u M ) was injected intracerebroventricularly (icv) 30 min prior to L P S injection. Scale bar, 25 um. D: The bar graph shows the number of OX-42 ir cells after P B S , L P S or L P S plus o x A T P injection (n=5 animals). *P < 0.05 compared to PBS-injected group, **P < 0.05 compared to LPS-injected group. 69 2.3.4 Effects of oxATP on LPS-induced Activation of p38 M A P K and N F K B L P S induces phosphorylation of p38 mitogen activated protein kinase (p38 M A P K ) , an intracellular signaling molecule involved in microglial activation (Xie et al., 2004). We have investigated the expression of p38 M A P K dependence on P 2 X 7 R in L P S -injected striatum (at 5 |ag for 6, 12, 24, 72 hrs) in the absence and presence of o x A T P (at 1 u M , injected 30 min prior to L P S injection). Little or no activation of p38 M A P K was observed in PBS-injected striatum (Figure 2-4A, upper panel). However, L P S injection (5 ug for 24 hr) elicited a large degree of phosphorylation of p38 M A P K (green) in OX-42 (+)ve microglia (red) (Figure 2-4B, upper panel). The injection of o x A T P effectively attenuated phosphorylation of p38 M A P K (Figure 2-4C, upper panel). The bar graph in Figure 2-4D (n=4 animals) shows the time-dependent changes in p38 M A P K phosphorylation following L P S injection and subsequent inhibition in the presence of o x A T P . Overall, at 24 hr, 42.2 ± 14.2 cells/mm 2 expressed phosphorylated p38 M A P K in PBS-injected striatum. The corresponding values in LPS-injected and L P S plus oxATP-injected striatum were 184.5 ± 17.4 cells/mm 2 and 112.4 ± 16.3 cells/mm 2 , respectively. The number of p38 M A P K (+)ve cells was significantly decreased in the presence of o x A T P by 39.1% compared to L P S alone. The corresponding decreases in p38 M A P K with o x A T P at 12 hr and 72 hr were 32.7% and 57.2%, respectively; all effects were significant. These results suggest that activation of P 2 X 7 R is linked with downstream p38 M A P K activity in microglia. The translocation of nuclear factor kappa B ( N F K B ) to the nucleus initiates transcription of a number of pro-inflammatory mediators such as T N F - a (Combs et al., 2001). We next studied expression of N F K B in microglia with L P S stimulation (at 5 ug 70 for 6, 12, 24, 72 hrs) in the absence and presence of o x A T P (at 1 u M , injected 30 min prior to L P S injection). Representative immunostaining showed little expression of N F K B in PBS-injected striatum (Figure 2-4A, lower panel). Intrastriatal injection of L P S (same as for p38 M A P K ) significantly upregulated nuclear translocation of the p65 subunit, a component of the N F K B p65/p50 heterodimer (Figure 2-4B, lower panel, green) localized with OX-42 (+)ve microglia (red). The expression of N F K B was considerably reduced in the presence of o x A T P (Figure 2-4C, lower panel). The bar graph in Figure 2-4E (n=4 animals) shows LPS-induced time-dependent increases in N F K B and inhibition in the presence of o x A T P . Overall, at 24 hr, 73.4 ± 19.8 cells/mm were expressed nuclear translocation of the p65 subunit in PBS-injected striatum. The corresponding values in LPS-injected and L P S plus oxATP-injected striatum were 403.2 + 42.1 cells/mm 2 and 264.1 + 35.2 cells/mm 2 , respectively. The number of p65 (+)ve cells were significantly decreased in the presence of o x A T P (by 34.5%) compared to L P S alone. A t the time points of 12 hr and 72 hr, ox A T P reduced N F K B by 33.8% and 46.1%, respectively, with both values reaching significance. Our results show inhibition of P 2 X 7 R attenuates LPS-induced activation of N F K B in microglia. 71 Figure 2-4. OxATP inhibits activation of p38 M A P K and N F K B (p65 subunit) in LPS-injected rat striatum Brain sections were double immunostained with phospho-p38 M A P K and OX-42 antibodies 24 hr post-injection with P B S (A, upper panel), L P S ( B , upper panel) and L P S plus o x A T P (C, upper panel). Brain sections were double immunostained with p65 and OX-42 antibodies 24 hr post-injection with P B S (A, lower panel), L P S ( B , lower panel) and L P S plus o x A T P (C, lower panel). O x A T P (at l u M ) was injected icv 30 min prior to L P S injection. Scale bars, 40 um. D : The bar graph shows the time course of L P S -mediated p38 M A P K activation in the absence or presence of o x A T P (n=4 animals). E : The bar graph shows the time course of LPS-mediated p65 activation in the absence or presence of o x A T P (n=4 animals). *P < 0.05 compared to PBS-injected group, **P < compared to LPS-injected group. 72 P B S L P S L P S + o x A T P 2.3.5 Effects of oxATP on LPS-induced iNOS Expression Microgl ial i NOS has been reported to contribute to cerebral ischemic injury in LPS-injected rat brain (Lee et al., 2005). The involvement of P 2 X 7 R in i N O S signaling was next investigated using R T - P C R and immunohistochemical analysis. Production of i N O S was observed in a small population of cells in PBS-injected brain (Figure 2-5 A ) but was considerably elevated with L P S injection (5 p,g at 24 hr post-injection) (Figure 2-5B). The inset of Figure 2-5B shows co-localization of i N O S (green) with OX-42 (+)ve microglia (red). In the presence o f o x A T P (at 1 u M , injected 30 min prior to L P S injection), LPS-induced production of i N O S was attenuated (Figure 2-5C). The time-dependent changes in numbers of i N O S (+)ve cells were measured at different time points (6, 12, 24, 72 hrs) with P B S injection or with L P S injection in the absence and presence of o x A T P (at 1 uM) . A s shown in Figure 2-5D (n=4 animals), little or no i N O S production was measured at any time point in PBS-injected rat brain. With L P S injection, the number of i N O S (+)ve cells reached a maximum at 24 hr and returned to a basal level at 72 hr post-injection. The numbers of i N O S (+)ve cells were significantly reduced at 12 hr (43.3% reduction), and 24 hr (56.3% reduction) in the presence of ox A T P (at 1 uM) compared to L P S alone. R T - P C R was used to determine i N O S m R N A expression in P B S , L P S and L P S plus ox A T P injected rat striatum. A representative R T - P C R result is presented in Figure 2-5E (upper panel) showing no expression of i N O S in PBS-injected rat striatum (lane 1). L P S (5 |ag, 12 hr post-injection) induced a high level of iNOS expression (lane 2) which was attenuated with o x A T P treatment (lane 3). Semi-quantification of R T - P C R results (n=3 experiments) are presented in Figure 2-5E (lower panel). L P S injection induced a 6-74 fold increase of i N O S expression relative to PBS-injected rat striatum and L P S plus o x A T P significantly reduced i N O S expression (by 67%) from that measured with L P S alone. We conclude that inhibition of P 2 X 7 R reduces LPS-induced expression and production of i N O S in rat striatum. 75 Figure 2-5. OxATP attenuates LPS-mediated iNOS expression in rat striatum Brain tissues were immunostained with iNOS antibody post-injection with P B S (A), L P S (B) and L P S plus o x A T P (C). L P S was injected (5 p.g for 24 hr) into rat striatum and o x A T P (at l u M ) was injected icv 30 min prior to L P S injection. Inset of B shows co-localization of i N O S and OX-42 (+)ve microglia. Scale bar, 30 urn. D: The bar graph shows the time course of i N O S expression reaching maximum 24 hr post-LPS injection. (n=4 animals). E : R T - P C R results for iNOS expression are presented from LPS-injected (12 hr) rat striatum (n=3 animals), (i-actin used as a reaction standard. The bar graph shows semi-quantitative R T - P C R results for expression of i N O S in rat striatum. *.P<0.05 compared to PBS-injected group, **P < 0.05 compared to LPS-injected group. 76 2.3.6 Effects of oxATP on LPS-mediated Nitration The enhancement of i N O S with L P S injection prompted us to examine the involvement of P 2 X 7 R on LPS-induced nitration of proteins using 3-nitrotyrosine (3-NT) immunostaining. Peroxynitrite, formed from superoxide anion and N O , induces nitration of tyrosine residues on proteins and produces 3-NT (Pryor and Squadrito, 1995). 3-NT has been documented as a primary marker for oxidative stress induced nitration (Wu et al., 2002). Representative immunostaining results indicate that little or no 3-NT (+)ve cells were evident in PBS-injected control (Figure 2-6A, upper panel). However, L P S injection (5 |ag, 3 d post-injection) increased the number of 3-NT (+)ve cells (Figure 2-6B, upper panel). In the presence of o x A T P (at 1 u M , injected 30 min prior to L P S injection) with L P S , the number of 3-NT (+)ve cells was markedly attenuated (Figure 2-6C, upper panel). Double staining immunofluorescence was then used to determine association of 3-N T with microglia. The results (Figure 2-6 (A-C) , lower panels) showed a high degree of co-localization of OX-42 (+)ve microglia with 3-NT (+)ve cells in LPS-injected rat striatum. The presence of o x A T P with L P S inhibited both OX-42 and 3-NT ir (Figure 2-6 (A-C) , lower panels). The bar graph summarizing results is presented in Figure 2-6D (n=5 animals). Overall, a low level of 3-NT (+)ve cells were co-localized with microglia in PBS-injected striatum (1.1 + 0.6/mm 2 cells). The corresponding values in LPS-injected and L P S plus oxATP-injected striatum were 40.1 ± 4.8/mm 2 cells and 21.9 ± 2.1/mm 2 cells, respectively. The number of 3-NT (+)ve cells was decreased in the presence of o x A T P by 45.3% compared to L P S alone. These findings suggest a role for P 2 X 7 R in 77 microglia in mediating cellular production of peroxynitrite and oxidative stress in L P S -injected striatum. 78 H Z i •Of X CD A C B 9 P B S L P S L P S + o x A T P D 50 40 30 13 o _m 20 -H z m 10 0 ** P B S LPS L P S + o x A T P Figure 2-6. OxATP reduces LPS-mediated nitration of proteins Brain tissues were immunostained with 3-NT antibody 3 d post-injection with P B S (A, upper panel), L P S (B, upper panel) and L P S plus o x A T P (C, upper panel). Brain sections were also double immunostained with 3-NT and OX-42 antibodies 3 d post-injection with P B S (A, lower panel), L P S (B, lower panel) and L P S plus o x A T P (C, lower panel). O x A T P (at l u M ) was injected icv 30 min prior to L P S injection. Scale bars, 40 um (top panel), 20 um (bottom panel). D: The bar graph shows effects of o x A T P on L P S -mediated increase of 3-NT ir microglia (n=5 animals). *P < 0.05 compared to P B S -injected group, **P < compared to LPS-injected group. 79 2.3.7 Effects of oxATP on LPS-mediated Lipid Peroxidation and Oxidative DNA Damage Lip id peroxidation is an autocatalytic process commenced by reactive oxygen species (ROS) generated by cellular oxidative stress. 4-Hydroxynonenal (4-HNE) is a major end product of l ipid peroxidation and used as a marker for lipid peroxidation (Ejima et al., 2000). We have studied the effect of P 2 X 7 R on LPS-induced lipid peroxidation (4-HNE marker) in the rat striatum. L P S injection (5 [ig for 24 hr) induced extensive 4 - H N E ir indicating l ipid peroxidation in the striatum compared to PBS-injected striatum (Figure 2-7A, upper panels). O x A T P (at 1 u M , injected 30 min prior to L P S injection, upper right panel) was effective in reducing the 4 - H N E (+)ve areas compared to LPS-injected striatum. Quantification in levels of 4 - H N E are presented in Figure 2-7B with L P S causing a considerable increase in 4 - H N E ir (41 fold increase vs. PBS) . Treatment with o x A T P was effective in reducing 4 - H N E (by 50% vs. L P S alone; n=5 animals). Reactive oxygen species (ROS) can act on D N A causing breakdown of D N A strands and base modification. 8-OHdG is an oxidized form of deoxyguanosine (dG), one of the constituents of D N A used as a marker for oxidative D N A damage (Kasai et al., 1987). Representative results are presented showing L P S (5 jag for 24 hr), but not P B S , injection was associated with 8-OHdG ir and oxidative D N A damage (Figure 2-7A, lower left and middle panels). In the presence of o x A T P (at l u M , injected 30 min prior to L P S injection), LPS-induced oxidative D N A damage was reduced (Figure 2-7A, lower right panel). Quantification of data is presented in Figure 2-7C with L P S injection eliciting a large increase in 8-OHdG ir (52 fold increase vs. PBS) . L P S plus o x A T P treatment . 8 0 significantly reduced 8-OHdG (+)ve areas by 54% compared to LPS-injected striatum (n=5 animals). Our results suggest that P 2 X 7 R is linked with oxidative stress and that o x A T P inhibition of this purinergic subtype receptor reduces l ipid peroxidation and oxidative D N A damage in inflamed microglia. 81 Figure 2-7. OxATP reduces LPS-induced lipid peroxidation and oxidative DNA damage A : Brain tissues were immunostained with 4 - H N E antibody 24 hr post-injection with PBS (upper left panel), L P S (upper middle panel) and L P S plus o x A T P (upper right panel). Brain sections were immunostained with 8-OHdG antibody 24 hr post-injection with P B S (lower left panel), L P S (lower middle panel) and L P S plus o x A T P (lower right panel). O x A T P (at 1 uM) was injected icv 30 min prior to L P S injection. Scale bar, 1.0 mm. Bar graphs show L P S stimulation of 4 - H N E and 8-OHdG and effects of o x A T P to block LPS-induced l ipid peroxidation and oxidative D N A damage (B and C, respectively) (n=5 animals). *P < 0.05 compared to PBS-injected group, **P < compared to L P S -injected group. 82 2.3.8 Effects of oxATP on LPS-induced Neuronal Caspase-3 Activation Increase in caspase-3 activity has been reported in LPS-induced neuronal cell death in co-cultures of neurons and glial cells (Lee et al., 2G04) and in LPS-injected animal models of neurodegeneration (Cunningham et al., 2005). We have investigated the effects of o x A T P on LPS-mediated neuronal caspase-3 activation at 24 hr post-LPS injection. Representative staining results for caspase-3 (green) and neurons (NeuN, red) are presented in Figure 2-8 (A-C) . The results show an absence of caspase-3 in P B S -injected striatum (Figure 2-8A) and elevated caspase-3 in LPS-injected (Figure 2-8B, at 24 hr post-injection). Pretreatment with o x A T P (at 1 u M , injected 30 min prior to L P S injection) reduced caspase-3 activation in LPS-injected brain (Figure 2-8C). We examined caspase-3 ir at different time points (6, 12, 24, 72 hrs) of L P S post-injection and found a maximum caspase-3 activation at 24 hr with reduced levels at 72 hr (Figure 2-8D, n=4 animals). The effects of o x A T P on LPS-mediated neuronal caspase-3 activation are presented in Figure 2-8E. At 24 hr post-LPS injection, 18.1 ± 3.4 cells/mm 2 were caspase-3 (+)ve and o x A T P at 1 u M was effective in inhibiting (50% vs. L P S alone) the activation of neuronal caspase-3 (n=4 animals). These results suggest that LPS-induced activation of P 2 X 7 R in microglia may in part be involved in activation of neuronal caspase-3 leading to neuronal loss in LPS-injected animals. 83 I" is SB :-| w A B C 1 T» P B S L P S L P S + o x A T P D 25 i S20 o i is ^ 10 <l> i 5 O 0 E i l l P B S 6h 12h 24h 72h PBS L P S LPS+oxATP Figure 2-8. Neuronal caspase-3 is activated in a time-dependent manner and is inhibited by blocking P2X7R NeuN (+)ve cells are presented in red and caspase-3 shown in green. Brain sections were double immunostained with caspase-3 and N e u N antibodies 24 hr post-injection with P B S (A), L P S (B) and L P S plus o x A T P (C). O x A T P (at 1 uM) was injected icv 30 min prior to L P S injection. Scale bar, 15 urn. D: L P S injection induced a time-dependent increase of caspase-3 (+)ve neurons reaching its maximum at 24 hr (n=4 animals). E : The bar graph shows effects of o x A T P on LPS-mediated (at 24 hr) increase of caspase-3 ir neurons (n=4 animals). *P < 0.05 compared to PBS-injected group, **P < compared to LPS-injected group. 84 2.3.9 Effects of oxATP on LPS-induced Neuronal Loss A n important aspect of this work was to determine effects of P 2 X 7 R modulation on extents of neuronal damage. We have studied this point by measuring neuronal viability in LPS-injected striatum (5 \ig for 3 d) in the absence and presence of ox A T P (at 1 u M , injected 30 min prior to L P S injection). Neuronal viability was assessed by counting immunostained neurons using neuron-specific nuclear protein (NeuN) antibody (Dou et al., 2003; R y u et al., 2004). A typical result is shown in the panels of Figure 2-9 (A-C) . The number of N e u N (+)ve cells was considerably decreased in LPS-injected striatum (Figure 2-9B) compared to PBS-injected control (Figure 2-9A). However, in the presence of o x A T P treatment, numbers of N e u N (+)ve cells were increased compared to LPS-injected striatum (Figure 2-9B,C). The overall results are shown in the bar graph in Figure 2-9D (n=5 animals). Numbers of N e u N (+)ve cells were counted in PBS-injected striatum and used as normalized control (100%) to analyze neuronal viability. N e u N (+)ve cells were significantly decreased to 39.5 ± 1.8% in LPS-injected striatum compared to P B S -injected striatum. However, ox A T P pretreatment significantly increased numbers of striatal neurons to a level of 62%> representing a significant degree of neuroprotection. Thus, activation of P 2 X 7 R in microglia in inflammatory conditions is associated with enhancement of neuronal damage, an effect which is partially inhibited in the presence of ox A T P . 85 D Figure 2-9. OxATP increases neuronal viability in LPS-injected rat brain (3 d post-LPS injection) Brain sections were immunostained with NeuN antibody 3 d post-injection with PBS (A), L P S (B) and L P S plus o x A T P (C). O x A T P (at 1 uM) was injected icv 30 min prior to L P S injection. Scale bar, 50 ^m. D: The bar graph shows effects of o x A T P on L P S -mediated decrease of N e u N ir (n=5 animals). *P < 0.05 compared to PBS-injected group, **P < compared to LPS-injected group. 86 2.3.10 Effects of oxATP on LPS-induced Gene Expressions in Cultured Human Microglia L P S induces a number of inflammatory mediators in human microglia (Nagai et al., 2001; Choi et al., 2002). We have examined the involvement of P 2 X 7 R in L P S -induced gene expressions for pro- and anti-inflammatory mediators and chemokines in human fetal microglia using R T - P C R . We first determined that exposure of cultured microglia to L P S (100 ng/ml) increased expression of P 2 X 7 R in a time-dependent manner (Figure 2-1 OA). A low constitutive expression of receptor was detected in unstimulated microglia with maximum levels found at 8 hr after L P S treatment. In the presence of o x A T P (at 300 u M , applied 2 hr prior to L P S treatment), LPS-induced upregulation o f P 2 X 7 R was attenuated to near basal level (Figure 2-10B). Microgl ia were treated with L P S for 8 hr, in the absence and presence of o x A T P (at 300 u M , applied 2 hr prior to L P S treatment), to examine expressions of inflammatory mediators. A representative result is presented in Figure 2-10C. The results show no basal expressions of T N F - a , IL-6, I L - i p , IL-12 or C O X - 2 in control (Figure 2-10C, lane 1). A l l inflammatory mediators were markedly expressed following human microglia exposure to L P S (Figure 2-10C, lane 2). O x A T P abolished expressions of all factors (Figure 2-10C, lane 3). o x A T P treatment alone had no effect to alter expressions of the pro-inflammatory mediators (Figure 2-10C, lanes 4). We next examined effects of L P S and o x A T P on expression of the chemokines IL-8 and M C P - 1 and the anti-inflammatory cytokines T G F - p i and IL-10. L P S stimulation of human microglia induced expression of both chemokines which were absent in unstimulated cells (Figure 2-10D). O x A T P treatment partially reduced expression of M C P - 1 but not IL-8. Both TGF-P 1 and IL-10 were constitutively expressed 87 in unstimulated microglia and exposure to L P S did not alter the expression of these anti-inflammatory cytokines. Semi-quantitative R T - P C R results for pro- and anti-inflammatory cytokines and the chemokines are presented in the bar graphs in Figure 2-10E, F (n=3 experiments). o x A T P was effective in reducing band intensities to below detectable levels for all pro-inflammatory mediators (Figure 2-10E). Block of P 2 X 7 R reduced L P S induction of M C P - 1 expression by 50% with no effects on IL-8 or anti-inflammatory cytokines (Figure 2-1 OF). The R T - P C R results indicate that inhibition of P 2 X 7 R using o x A T P inhibits expression of. pro-, but not anti-, inflammatory mediators in L P S stimulated human microglia. 88 Figure 2-10. OxATP blocks LPS-induced pro-inflammatory mediators, but not anti-inflammatory cytokines in cultured human microglia A : The expression of P2X7R were measured using R T - P C R , microglia were treated with L P S (100 ng/ml) at different time points (0, 1, 4, 8, 24, 48 hrs). B: L P S (100 ng/ml for 8 hr) induced upregulation of P 2 X 7 R expression, which was inhibited by o x A T P (at 300 u M , applied 2 hr prior to L P S treatment). C: L P S (100 ng/ml for 8 hr) treatment induced upregulation of pro-inflammatory mediators including T N F - a , C O X - 2 , IL-6, I L - i p and IL-12; all factors were inhibited by o x A T P (at 300 u M , applied 2 hr prior to L P S treatment). D: LPS-induced expression of M C P - 1 , but not IL-8, was attenuated by o x A T P . L P S did not alter expression of anti-inflammatory cytokines, IL-10 and TGF-P 1. G A P D H was used as a reaction standard. E : Semi-quantitative R T - P C R results for pro-inflammatory mediators are shown as bar graph (n=3 experiments). F: Semi-quantitative R T - P C R results for chemokines and anti-inflammatory cytokiness are presented (n=3 experiments). *P < 0.05 compared to PBS-treated group, **P < compared to LPS-treated group. 89 4 LPS(100ng/ml) Oh lh 4h 8h 24h 48h 0 ° V V P 2 X 7 R P 2 X 7 R Cr v V o D G° V V o E T N F - a C O X - 2 IL-6 I L - l p IL-12 • PBS • LPS • LPS + oxATP • oxATP a; > < E • r—t 2.5 2.0 1.5 1.0 0.5 0.0 1PBS • LPS ILPS + oxATP • oxATP IL-8 M C P - 1 IL-10 TGF-P 1 2.3.11 Effects of oxATP on LPS-modulated [Ca2 +]i in Cultured Human Microglia L P S treatment elevates intracellular C a 2 + levels [Ca 2 + ]i in microglia (Choi et al., 2002; Hoffmann et al., 2003). Changes in [Ca 2 +]j have been linked with altered cellular functional responses including gene expression and proliferation (Hide et a l , 2000; Choi et al., 2002; Hooper et al., 2005). We initially measured effects of the selective agonist of P2X7R, B z A T P , to mobilize [Ca 2 +]j in human microglia. B z A T P (300 uM) application (control) induced a prolonged [Ca 2 + ]i increase which was abolished with o x A T P pretreatment (at 300 u M , applied 2 hr prior to B z A T P application) (Figure 2-11 A ) . In the absence of extracellular C a 2 + , B z A T P had no effect to alter [Ca 2 + ]i (Figure 2-11 A ) . The importance of C a 2 + entry was also demonstrated using M n 2 + quenching experiments. A s shown in Figure 2-1 I B , administration of B z A T P in the presence of M n 2 + caused a rapid loss of fluorescence (measured at 360 nm) indicating the nature of an influx pathway. The C a 2 + ionophore, ionomycin, elicited a small additional quenching of fluorescence as previously found 9+ using other stimuli for C a influx in microglia (Wang etal., 1999). Additional studies investigated possible interactions between B z A T P and L P S in modulating mobilization of [Ca 2 + ] i in human microglia. Microgl ia were exposed to L P S (100 ng/ml for 2 hr) prior to calcium imaging experiments. A typical sustained B z A T P (300 uM) response is presented in Figure 2-11C (labeled as control, middle trace). In the presence of L P S pretreatment (100 ng/ml for 2hr), the BzATP-induced increase in [Ca 2 + ]i was considerably enhanced relative to control (Figure 2-11C, upper trace). The potentiation by L P S of the B z A T P response was blocked by o x A T P (at 300 u M , applied 2 hr prior to L P S treatment, lower trace, Figure 2-11C). 91 The bar graph summarizes results from experiments using procedures of Figure 2-11C is presented in Figure 2-1 I D showing a significant increase of peak amplitudes (by 274%) of [Ca ]i with L P S pretreatment compared to B z A T P response in the absence of 9-4-L P S (n=5 separate experiments). BzATP-induced enhancement of [Ca ]i in the presence of L P S (100 ng/ml for 2 hr) was reduced with pretreatment of o x A T P (by 89%). These results suggest that L P S potentiation of P2X 7 R-mediated [Ca 2 + ] i mobilization may lead to modulation of functional responses in human microglia. 92 1.2-, BzATP 300nM 1.0 1 Time (sec) Figure 2-11. LPS pretreatment modulates P2X ? R mediated [Ca2+]j mobilization in cultured human microglia A : B z A T P (at 300 uM) induced a prolonged [Ca 2 +]j increase (upper trace; n=22 cells) blocked by o x A T P pretreatment (at 300 u M , applied 2 hr prior to L P S treatment, middle trace; n=16 cells). B z A T P induced a minimal increase of [Ca 2 +]j in the absence o f extracellular C a 2 + (lower trace; n=23 cells). B: M n 2 + quenching shows B z A T P application was associated with a significant decrease in fluorescence indicative of [Ca2"1"^ influx in human microglia (n=20 cells). C: L P S (for 2 hr at 100 ng/ml, n=28 cells, upper trace) pretreatment significantly enhanced B z A T P to increase [Ca 2 +]j compared to control (cells with no L P S exposure, n=25 cells, middle trace). O x A T P (at 300 u M , pretreated for 2 hr prior to L P S treatment, n=33 cells, lower trace) preincubation inhibited L P S potentiation of B z A T P to increase [Ca 2 +]j in cultured human microglia. D: the bar graph shows L P S potentiation of B z A T P to increase [Ca 2 + ] i which was inhibited by o x A T P . n=5 independent experiments carried out for all calcium experiments. *P < 0.05 compared to control group (no exposure to LPS) , **P < compared to L P S pretreated group. 93 2.4 DISCUSSION The important finding from this work is that activation o f P 2 X 7 R in LPS-injected rat striatum is linked to enhancement of a host of inflammatory responses and mediators including microgliosis, levels of i N O S , oxidative stress, intracellular signaling factors p38 M A P K and N F K B and neuronal viability. Importantly, o x A T P was effective in the inhibition of LPS-induced inflammation and was neuroprotective in inflamed brains. A s discussed below, the overall results from in vivo and in vitro studies implicate P 2 X 7 R in microglia in mediating purinergic inflammatory responses. L P S induced the expression of P 2 X 7 R in a time-dependent manner following intrastriatal injection. The ionotropic receptor was maximally expressed at 12 hr post-L P S injection; little or.no P 2 X 7 R ir was evident under basal conditions or at 48 hr after L P S . Double staining immunofluorescence study showed P 2 X 7 R to be primarily associated with microglia with a smaller proportion of purinergic receptor co-localized with astrocytes; N e u N (+)ve cells showed no expression of P 2 X 7 R (Figure 2-2). Both types of glial cells showed significant increases in P 2 X 7 R ir for L P S , compared to P B S , injection, however, only microglial-mediated responses were further studied. Previous work has documented P 2 X 7 R in glia (Collo et al., 1997) with some reports for the presence of this purinergic receptor subtype in neurons (Deuchars et al., 2001; Franke et al., 2004). Immunostaining patterns for OX-42 (+)ve microglia (Figure 2-3A,B) indicated a predominantly ramified morphology for P B S injection with cells showing small bodies and fine processes. In contrast, L P S intrastriatal injection was associated with swollen cell bodies and retracted processes. These results indicate L P S injection caused activation 94 of microglia, as indicated by a shift in morphology from ramified to ameboid, and also a proliferative response (Figure 2-3D). O x A T P pretreatment prior to L P S injection was effective in reducing numbers of activated microglia with the OX-42 (+)ve cells mainly exhibiting an ameboid morphology (Figure 2-3C). This result would suggest that o x A T P reduced the number of activated microglia but had little effect to block cellular activation. Supranigral L P S administration has been reported to increase numbers of OX-42 (+)ve microglia and to induce a shift in morphology from resting to activated state (measured at 24 hr post-LPS injection) (Iravani et al., 2005). However, at a longer time of 30 d post LPS-injection, numbers of OX-42 (+)ve microglia were markedly reduced with cells showing a predominant ramified morphology. Bianco et al (2006) have reported that decrease of P 2 X 7 R was responsible for LPS-induced inhibition of proliferative responses in N 9 and rat primary microglia. Down-regulation of P 2 X 7 R expression using R N A interference or P 2 X 7 R deficient clones decreased LPS-induced inhibition of microglial proliferation. Double immunofluorescence study showed LPS-induced similar patterns of time-dependent changes in the intracellular signaling pathway p38 M A P K and the nuclear transcription factor N F K B in microglia (Figure 2-4). Both responses were maximal at 24 hr post-LPS and remained elevated at 3 d; values for both p38 M A P K and N F K B were relatively constant and low over the measurement period for P B S injection. Treatment with ox A T P effectively reduced ir for both p38 M A P K and N F K B for all but the earliest time point of 6 hr post-LPS injection. The similarity in time dependent changes and pharmacological actions of o x A T P would support coupling between the two intracellular inflammatory mediators. Taken together with the results from Figure 2-3, the 95 inflammatory pathway p38 M A P K and factor N F K B appear to regulate a proliferative microglial response rather than cellular activation. Proliferation of microglia has been associated with phosphorylation of p38 M A P K (Tikka et al., 2001) and activation of N F K B (Ryu et al., 2002; Suo et al., 2003). Considerable evidence has been accumulated suggesting nitrogen species mediate responses in inflamed brain (Brown and Bal-Price, 2003; Lee et a l , 2005). This point was examined using immunohistochemical procedures to determine levels of inducible enzyme i N O S and 3-NT. Representative staining showed marked upregulation of i N O S in LPS-injected striatum (at 24 hr) with co-localization to microglia (Figure 2-5); in the presence of o x A T P , i N O S levels were significantly reduced. R T - P C R results (at 12 hr post-LPS injection) also indicated upregulation of the enzyme relative to control (PBS injection) and sensitivity to o x A T P . The present results are consistent with the finding of o x A T P effects to downregulate iNOS expression and N O production in LPS-primed R A W macrophages (Hu et al., 1998). The inhibition of i N O S has a reported neuroprotective effect in an animal model of LPS-mediated inflammation (Arimoto and Bing, 2003). Double immunofluorescence staining, carried out at the time of maximum i N O S response (24 hr post-LPS injection), indicated co-localization of 3-NT with OX-42 (+)ve microglia (Figure 2-6 (A-C)) . The expressions of 3-NT and OX-42 markers were attenuated by o x A T P . The diminished 3-NT ir in microglia in response to o x A T P injection would be consistent with effects of the P 2 X 7 R inhibitor to reduce the activity of i N O S . The involvement of 3-NT in LPS-induced brain damage (Tomas-Camardiel et al., 2004) and in Alzheimer's disease (AD) brain (Hensley et al., 1998; Castegna et al., 2003) 96 have been documented. Recent work has also reported glial-derived nitrogen species mediate enhanced blood brain barrier ( B B B ) permeability in amyloid-beta injected rat hippocampus (Ryu and McLarnon, 2006). Oxidative stress is characterized by increased levels of l ipid peroxidation (4-HNE marker) and D N A damage (8-OHdG marker): Both markers were absent in PBS-injected striatum, considerably elevated with L P S injection and reduced with o x A T P pretreatment of LPS-injected striatum (Figure 2-7). In the latter case, similar effects o f o x A T P to block 4 - H N E (by 56%) and 8-OHdG (by 60%), relative to L P S alone, were measured. The effects of o x A T P to inhibit l ipid peroxidation is a novel finding. Elevated 4 - H N E levels are observed in A D brain and in cerebral ischemia (McGrath et al., 2001; McKracken et al., 2001). A number of stimuli, including L P S , activate neuronal caspase-3 leading to cellular apoptosis (Deng et al., 2004; Minogue et al., 2003). Double immunostaining (NeuN/caspase-3) showed upregulation of neuronal caspase-3 in LPS-injected striatum with suppression by o x A T P . To translate caspase-3 activity to neuronal viability, numbers of N e u N (+)ve striatal neurons were counted. A t 3 d post-LPS injection, numbers of neurons were reduced relative to P B S with partial recovery for o x A T P pretreatment prior to L P S application. The involvement of caspase-3 in neuronal damage (Figure 2-8) is consistent with findings of upregulation of this factor in L P S (Cunningham et a l , 2005) and quinolinic acid (Ryu et al., 2005)-injected rat brain. In the latter study, pharmacological inhibition of microglial activation reduced neuronal caspase-3 activation and was neuroprotective. 97 We were interested i f direct L P S stimulation of cultured human microglia would support the in vivo findings. L P S induced P 2 X 7 R expression in cells with partial block by o x A T P (Figure 2-10A,B) indicating that o x A T P interferes with a signal to increase m R N A . L P S stimulation of microglia elevated expressions of pro-inflammatory factors ( C O X - 2 , T N F - a , IL-6, IL-1(5, IL-12) which was blocked by o x A T P pretreatment prior to L P S . Expressions of the chemokines IL-8 and M C P - 1 were increased by L P S , however, only M C P - 1 was sensitive to o x A T P . L P S stimulation had no effect to alter expressions of the anti-inflammatory cytokines IL-10 and TGF-P 1. The in vitro data strongly suggests that stimulation of P2X7R in microglia leads to enhanced cellular expression of an assemblage of pro-inflammatory mediators which, in total, cause neuronal damage. A possible mechanism to account for P 2 X 7 R mediated actions is increased cellular entry of C a . Stimulation of P 2 X 7 R with the specific purinergic ligand B z A T P caused a sustained increase in [Ca 2 + ]i which was blocked by o x A T P and abolished in Ca -free PSS. A novel finding was that L P S pretreatment was highly effective in the potentiation of [Ca 2 + ]i induced by B z A T P (Figure 2 - l l C , D ) . This result would be consistent with direct L P S enhancement in numbers of P 2 X 7 R or LPS-induced microglial release of A T P which could activate P 2 X 7 receptors (see Figure 2-12). High levels of A T P (likely in excess of 1 m M ) are required to activate P 2 X 7 R (Hide et al., 2000; McLarnon, 2005). Elevated brain concentrations of A T P would only be expected under abnormal situations whereby endogenous' cells were damaged and chronically leaky. High levels of A T P could then act as a chemoattractant to elicit a microglial proliferative response. 98 A schematic diagram is presented in Figure 2-12 depicting a putative P 2 X 7 R -mediated scheme linking neuronal damage to L P S stimulation. In vivo (Figure 2-1,2) and in vitro (Figure 2-10) results suggest direct effects of L P S to increase expression of P 2 X 7 R and microglial inflammatory responses. This possibility is supported by the reported presence of a highly conserved L P S binding motif for P 2 X 7 R (Denlinger et al., 2001, 2003). L P S could also cause cellular release of A T P as has been documented for microglia (Ferrari et al., 1997) and endothelial cells (Bodin and Burnstock, 1998; Sylte et al., 2005). Enhanced extracellular A T P activation of P 2 X 7 R in microglia could then lead to cellular secretion of inflammatory mediators such as R O S and pro-inflammatory cytokines. A s noted above, A T P is a potent chemotactic stimulus for microglial proliferative responses which could sustain an inflammatory environment and chronically elevated levels of A T P from stimulated and damaged cells. In conclusion, the present work has documented critical roles of P 2 X 7 R in an L P S animal model of inflammation. Some supporting evidence for involvement of C a 2 + influx mediated by this purinergic receptor subtype has been derived from in vitro study. Importantly, inhibition of P2X 7R-mediated signaling by o x A T P has been found effective in limiting inflammatory responses and conferring neuroprotection. Overall, pur results suggest modulation of P 2 X 7 R as a plausible strategy for therapeutic intervention of inflammation in neurodegenerative disease. 99 LPS I ATP • I • P2X 7R | oxATP [Ca2+]i p38 M A P K NFkB • Glial activation 02", NO, ONOO-Pro-inflammatory ^ f cytokines Neuronal damage (Lack of NeuN (+) cells, activation of Caspase-3) Figure 2-12. Schematic diagram depicting a putative P2X?R-mediated scheme linking neuronal damage to LPS stimulation In vivo and in vitro results suggest direct effects of L P S to increase expression of P 2 X ? R and microglial inflammatory responses. L P S could also cause cellular release of A T P . Enhanced extracellular A T P activation of P 2 X ? 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Blockade of microglial activation is neuroprotective in the l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine mouse model of Parkinson disease. J Neurosci 22(5): 1763-71. X i e Z , Smith C J , Van Eldik L J . 2004. Activated glia induce neuron death via M A P kinase signaling pathways involving J N K and p38. Gl ia 45(2): 170-9. 106 C H A P T E R 3: U P R E G U L A T E D EXPRESSION O F PURINERGIC P2X 7 R E C E P T O R IN A L Z H E I M E R ' S DISEASE AND A M Y L O I D - B E T A PEPTIDE T R E A T E D M I C R O G L I A AND IN PEPTIDE I N J E C T E D R A T HIPPOCAMPUS 2 3.1 INTRODUCTION Increased extracellular levels of A T P are associated with cellular damage in brain pathology. Leak of A T P from damaged cells serves as a chemotactic gradient and signal for mobilization and activation of microglia, the immune responding cells of the brain. Upon activation, microglia express and produce a diversity of factors which in assemblage constitute an inflammatory response to help resolve the original damage. However, this response has the potential of either aiding in the promoting of cell survival to repair injury or, in extreme cases, possibly exacerbating the neuronal damage. The net outcome of the inflammatory response is likely dependent on a number of factors including the local concentration of A T P and the duration and nature of the initial damage. Presumably, higher and chronic levels of A T P could be indicative of more severe cell damage thus favoring a pro-inflammatory response from microglia. Purinergic receptors comprise the metabotropic P 2 Y R family and the ionotropic P 2 X R family (Burnstock, 1996). The P 2 Y R are G-protein coupled to inositol triphosphate-mediated calcium (Ca ) release from endoplasmic reticulum (ER) stores. Depletion of C a 2 + from stores leads to a secondary entry of C a 2 + through store-operated channels (SOC) located in plasma membrane. The P 2 X R family comprises P 2 X i R -P 2 X 7 R and which are directly coupled to non-selective cationic channels allowing influx of N a + and C a 2 + , efflux of K + and a net cell depolarization. Functional interactions between the two subtypes of purinergic receptors have been reported in human microglia y 2 1 A version of this chapter has been submitted for publication. McLarnon JG, Ryu JK, Walker DG, Choi HB. Upregulated Expression of Purinergic P2X 7 Receptor in Alzheimer's Disease and Amyloid-Beta Peptide Treated Microglia and in Peptide Injected Rat Hippocampus. 107 with cell depolarization, mediated by A T P binding to a subtype P2XR (not P2X7R), 9+ acting to inhibit influx of Ca through S O C subsequent to A T P activation of P 2 Y R (Wang et al., 2000; McLarnon, 2005). A particularly unique member of P2XR is P2X7R which, when activated by A T P binding, forms a large pore allowing entry of large hydrophilic cationic molecules. The results from a number of studies have suggested that extracellular levels of A T P in excess of 1 m M are generally required for activation of P 2 X 7 R ( Hide et al., 2000; Moller , 2002; McLarnon, 2005). The high concentration of A T P required to open channels could indicate that functional responses mediated by P 2 X 7 R are associated with ongoing cellular damage and chronic brain inflammation. In this regard, activation of P 2 X 7 R in microglia has been correlated with production of the pro-inflammatory cytokines T N F - a (Hide et al., 2000) and I L - l p (Ferrari et al., 1997; Sanz and D i Virg i l io , 2000) and also with cell apoptosis (Ferrari et al, 1999; Brough et al., 2002). Recent work has documented roles of P 2 X 7 R in the microglial production of superoxide and that expression of the receptor is upregulated in a transgenic mouse model of A D (Parvathenani et al., 2003). In this study we have compared the expression of P 2 X 7 R in microglia obtained from neuropathologically-assessed A D brain (6 cases) and N D (non-demented, 7 cases). In addition, we have compared expression of this subtype ionotropic receptor between amyloid beta peptide (APi.42)-treated and untreated cultured human fetal microglia. Calcium responses induced by the P 2 X 7 R ligand B z A T P have also been determined in untreated microglia or cells previously exposed to APi_42. Finally, we have measured 108 expression of P2X7R in AP1.42 and P B S (phosphate buffered saline, control) injected rat hippocampus. 109 3.2 M A T E R I A L S AND M E T H O D S 3.2.1 Adult Human Microglia Adult brains were obtained post-mortem from individuals who have signed informed consent forms for brain donation to the Brain Donation Program of the Sun Health Research Institute (Lue et al., 1999). Neuropathological analysis was carried out on all donated brains with diagnosis of A D using criteria established in the Consortium to Establish a Registry for Alzheimer's disease ' (CERAD). Non-demented (ND) brain was characterized by plaque and tangle scores inconsistent with A D and a lack of clinical dementia in donor individuals. Postmortem intervals for isolation of N D and A D brains were 2.8 ± 0,5 hr (n=6 cases) and 2.1 ± 0.2 hr (n=7 cases), respectively; these times were not significantly different. Cultured adult human microglia were obtained from brain tissue following published procedures (Lue at al., 1996; McLarnon et al., 2005). Briefly, microglia were isolated from other glial cells by differential adherence and maintained in Dulbecco's modified Eagle medium ( D M E M ) with high glucose (5 mg/ml) containing 10% fetal bovine serum (FBS, Hyclone, Salt Lake City, U T ) and 50 p-g/ml gentamicin. Microgl ia were used in experiments after 10-14 days in culture and cell purity was assessed by using immunocytochemistry with antibodies to C D l i b (American Type Culture Collection [ A T C C ] , Manassas,VA), to Class II major histocompatibility antigen ( H L A -D R ) (1:800; I C N Pharmaceuticals, Irvine, C A ) and to CD68 (1:1000; Accurate Chemical and Scientific Corp. Westbury, N Y ) . Antibody to glial fibrillary acidic protein ( G F A P , 1:5,000; Dako, Raleigh, N C ) was used to measure astrocyte contamination. Immunocytochemical results showed purity of microglia was in excess of 98%. 110 3.2.2 Fetal Human Microglia Fetal cells were obtained from embryonic tissue following legalized therapeutic abortions as certified by the Ethics Committee of the University of British Columbia. The overall procedures of isolation and identification of microglia have been previously described (Satoh et al., 1995). In brief, brain tissues of 12-18 weeks gestation were incubated in phosphate buffered saline (PBS) with 0.25% trypsin and DNase I (40 p.g/ml) for 30 min at 37°C. Cells were separated into single cells by repeated gentle pipetting. Dissociated cells were then maintained in T75 flasks in D M E M containing 5% horse serum, 5 mg/ml glucose, 20 ug/ml gentamicin and 2.5 ug/ml amphotericin B . Floating microglia in a medium were harvested after 7-10 days and subsequently plated on six-well multiplates for R T - P C R . C D l l b and ricinus communis agglutinin ( R C A ) , specific markers for microglia, were used to confirm purity of the cultures exceeded 98%). 3.2.3 R T - P C R Analysis Human fetal microglia were plated on 6-well multiplates and were exposed to A P 1 . 4 2 or reverse peptide A P 4 2 - 1 (applications at 5 u M for 18 hr) or P B S . Adult microglia from N D individuals and A D patient were also used for R T - P C R experiments. Isolation of R N A s was performed using TRIzol (Gibco-BRL, Gaithersburg, M D ) and D N A contamination was eliminated using DNase. Complementary D N A (cDNA) synthesis was carried out using M - M L V reverse transcriptase (Gibco-BRL). For in vivo R T - P C R analysis, animals were deeply anesthetized and sacrificed by decapitation at 3 d post-injection of A P 1 . 4 2 , reverse peptide A P 4 2 - 1 or PBS . Following removal of brains, tissue samples from hippocampus were prepared and frozen in liquid 111 nitrogen. Total R N A was extracted using TRIzol subjected to DNase I treatment and c D N A synthesis carried out using M - M L V reverse transcriptase. M - M L V was omitted as a negative control. P C R primer sequences with expected product size are as follows: human P 2 X 7 R sense 5" - A C A A T G T T G A G A A A C G G A C T C T G A - 3 ' and human P 2 X 7 R antisense 5 " - C C G G C T G T T G G T G G A A T C C A C A T C - 3 " (728 bp); rat P 2 X 7 R sense 5'-A G G A G C C C C T T A T C A G C T C T - 3 ' and rat P 2 X 7 R antisense 5'-C A T T G G T G T A C T T G T C G T C C - 3 \ (692 bp); human G A P D H sense 5'-C C A T G T T C G T C A T G G G T G T G A A C C A-3" and human G A P D H antisense 5--G C C A G T A G A G G C A G G G A T G A T G T T C - 3 ' (251 bp); rat p-actin sense 5'-G T G G G G C G C C C C A G G C A C C A - 3 ' and rat p-actin antisense 5'-G T C C T T A A T G T C A C G C A C G A T T T C - 3 " (526 bp). P C R conditions were as follows: initial denaturation at 95°C for 6 min followed by 35 cycles of denaturation at 95°C for 45 s, annealing at 60°C for 1 min and extension at 72°C for 1 min. A final extension was carried out at 72°C for 10 min. The amplified-P C R products were identified using 1.5% agarose gels containing ethidium bromide and visualized under U V light. The intensities of each band were measured by densitometry using the N I H Image J 1.24 software (National Institutes of Health, Bethesda, M D , U S A ) and expressed as relative m R N A levels ( P 2 X 7 R m R N A levels normalized to G A P D H or P-actin) (Franciosi et al., 2005). Glyceraldehyde-3-phosphate dehydrogenase ( G A P D H ) and P-actin were used as reaction standards for human and rat, respectively. 112 3.2.4 Preparation and in vitro and in vivo Application of Amyloid Beta Peptide Full-length peptide (APi.42) was obtained from California Peptides, Napa, C A . Aggregated peptide for microglial stimulation was prepared following procedures outlined previously (Webster et al., 1997). Aggregated A P 1 . 4 2 was dissolved in 35% acetonitrile (Sigma, St. Louis, M O ) and diluted to 1.5 m M with sterile water. Sequential additions of P B S with vortexing of solutions were used to prepare a peptide concentration of 0.5 m M . A final step was to maintain peptide solution at 37 °C for 18 hr to enhance fibril formation and aggregation prior to storage at -20 °C. Reverse peptide AP42-1 (California Peptides) was also employed in some experiments with preparation following that described for A p i . 4 2 . Electron microscopy was used to confirm the aggregation of A P 1 . 4 2 in solution. Forward and reverse peptides were applied in vitro to cultured human fetal microglia. The studies used 18 hr exposure of cells to 5 u M concentrations of peptides. A similar treatment protocol has been applied to human microglia in testing for cellular expression and production of a spectrum of inflammatory mediators (Franciosi et al., 2005). For in vivo studies, male Sprague-Dawley rats (250-280 g, Charles River Laboratories) were anesthetized with intraperitoneal (i.p.) injection of a mixture of ketamine hydrochloride (72 mg/kg; Bimeda-MTC, Cambridge, Ontario, Canada) and xylazine hydrochloride (9 mg/kg; Bayer Inc., Etobicoke, Ontario, Canada) and then mounted in a stereotaxic apparatus (Kopf Instruments, Tujunga, C A ) . A P 1 . 4 2 or A p 4 2 - i ( lnmol in 2 ul) or P B S was slowly injected (0.2 ul/min) into the superior blade of dentate gyrus of the hippocampus (anteroposterior [AP]: -3.6 mm; mediolateral [ M L ] : -1.8 mm; dorsoventral [DV] : -3.2 mm) with a 10 ul Hamilton syringe. A l l animal procedures were 113 approved by the University of British Columbia Animal Care Ethics Committee, adhering to guidelines of the Canadian Council on Animal Care. 3.2.5 Calcium Sensitive Fluorescence Microscopy 94- 94-The procedures used for measurement of intracellular C a [Ca ]j have been documented (Choi et al., 2003). In brief, microglia were incubated with fura-2/AM (acetoxymethyl ester, at 1 u M , Molecular Probes, Eugene, OR) plus pluronic acid (at 1 u M ) in normal physiological saline solution (PSS) for 30 min. PSS contained (in m M ) : N a C l (126), KC1 (5), M g C l 2 ( 1 . 2 ) , H E P E S (10), D-glucose (10) and C a C l 2 ( l ) ; p H of 7.4. A l l reagents were obtained from Sigma (St. Louis, M O ) . Following a 20 min wash in dye-free PSS, coverslips were placed on the stage of a Zeiss Axiovert inverted microscope employing a x40 quartz objective lens. Cells were exposed to alternating excitation wavelengths of 340 nm and 380 nm at 6 s intervals and emission light was passed through a 510 nm filter. A n imaging system (Empix Imaging, Mississauga, O N , Canada) was used to record fluorescence ratios using a C C D camera (Retiga 1300i, Burnaby, B C , Canada). Results are presented as ratios F340/F380 vs time with all experiments done at room temperature (20-22°C). 3.2.6 Immunohistochemical Analysis Animals were deeply anesthetized and then transcardially perfused with heparinized cold saline followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (0.1 M P B , p H 7.4). The brains were removed from the skull and postfixed in the same fixative solution overnight and then placed in 30% sucrose for cryoprotection. The 114 brains were then frozen in powdered dry ice and stored at -70°C. Coronal sections throughout the hippocampus were cut at 40 urn intervals on a cryostat. The sections were stored in cryoprotectant solution. For P2X7R expression, free-floating sections were permeabilized with 0.2% Triton X-100 and 0.5% B S A in 0.1 M P B S for 30 min and then incubated overnight at 4 °C with primary antibody against P2X7R (1:200, Alomone Laboratory, Jerusalem, Israel). Sections were then sequentially incubated with Alexa Fluor-conjugated 488 anti-rabbit IgG (1:100; Molecular Probes) at R T for 2 hr in the dark. For double immunofluorescence immunohistochemistry, free-floating sections were incubated overnight at 4°C with a mixture of two primary antibodies: P 2 X 7 R in combination with OX-42 (1:200; Serotec, Oxford, U K ) or G F A P (1:500, Sigma). Sections were then incubated in a mixture of Alexa Fluor-conjugated 488 anti-rabbit IgG (1:100; Molecular Probes) and Alexa Fluor 594-conjugated anti-mouse IgG (1:100; Molecular Probes) at R T for 2 hr in the dark. Stained sections were examined under a Zeiss Axioplan 2 fluorescent microscope and images were acquired with a D V C camera (Diagnostic Instruments). For quantification of P 2 X 7 R immunoreactivity, mean gray level of immunoreactivity was measured and quantified using Northern Eclipse software (Empix Imaging, Mississauga, O N , Canada) (Ryu et al.,2004). A l l quantitative analyses were carried out in a blinded manner. 3.2.7 Statistical Analysis A l l results are presented as mean ± S E M . Statistical significance (P < 0.05) was evaluated using Student's Mest or one-way A N O V A , followed by Student-Newman-Keuls multiple comparison test where applicable (GraphPad Prism 3.0). 115 i 3.3 R E S U L T S 3.3.1 P2X7R Expression in Microglia from AD Patients and ND Individuals R T - P C R was carried out for expression of P 2 X 7 R in microglia obtained from 13 individuals. O f these, six were confirmed as A D and seven showed no diagnosis for A D symptoms and were confirmed as N D (see Materials and Methods). Representative band intensities for microglial P 2 X 7 R expressions are shown for one N D case and one A D case (Figure 3-1A); also shown is G A P D H as a reaction standard (lower panel, Figure 3-1A). In general, microglia obtained from N D individuals showed a weak band intensity for P 2 X 7 R indicating a low constitutive expression for this subtype purinergic receptor. However, microglia from all A D cases showed strong expressions for P 2 X 7 R. The results from semi-quantitative analysis of band intensities are presented in Figure 3-IB showing relative m R N A levels of P 2 X 7 R in A D and N D microglia. Overall, the expression of P 2 X 7 R was increased by 70% in A D microglia (n=6 cases) compared with N D cells (n=7 cases). 3.3.2 P2X 7 R Expression in APi_42-treated and Untreated Human Microglia A D brains are characterized by senile plaques of fibrillar A P i _ 4 2 peptide and neurofibrillary tangles. We next examined the effects of exposure of cultured human fetal microglia to A p V 4 2 (at 5 u M for 18 hr); two controls were used with one group treated with P B S and another group treated with reverse peptide A p 4 2 . i (at 5 u M for 18 hr). The representative results from a single R T - P C R assay for P 2 X 7 R are presented in Figure 3-1C. Human microglia exposed to P B S or to A p 4 2 . i exhibited a low level of band intensity 116 for P2X7R whereas A p Y 4 2 treated cells showed a robust expression for this subtype purinergic receptor (Figure 3-1C). The quantification of results is presented in Figure 3-1D. Treatment of human microglia with A P 1 . 4 2 peptide (n=6) significantly increased P2X7R expression (by 106%) relative to P B S control (n=6) and by 76% compared to reverse peptide (n=5). 117 Figure 3-1. Expression of P I X j R in N D and A D brain and in A P 1 4 2 stimulated human fetal microglia A : Typical R T - P C R for levels of P 2 X ? R in adult microglia obtained from N D and A D brain. G A P D H was used as a reaction standard. B: Relative m R N A levels for P 2 X 7 R from analysis of brains from 7 N D and 6 A D individuals. * P < 0.05 compared to N D . C : Representative expression of P 2 X 7 R with microglia exposed to P B S , A p 1 4 2 and A P 4 2 A with G A P D H used as a reaction standard. D : Relative expression of P 2 X 7 R for the different treatments (n= 6 for P B S and A P M 2 and n=5 for A P 4 2 , ) . * P < 0.05 compared to PBS treated group. ** P < 0.05 compared to A p t 4 2 treated group. 118 3.3.3 Functional Responses of P2X 7 R in APi_42 Pretreated and Untreated Human Microglia It was of interest to determine i f P 2 X 7 R mediated responses in human microglia were modulated by cellular exposure to A(3M2- This point was studied using the selective • 2+ P 2 X 7 R agonist B z A T P to elicit entry of Ca through the non-selective cationic channel associated with this receptor. Human microglial cells were placed in culture medium and grouped as control (no exposure to APi_4 2 peptide), APi.4 2-treated (exposure to 5 u M peptide for 18 hr) and APi.4 2-treated in the presence of ox A T P (a specific inhibitor of i P 2 X 7 R ) . In the latter group, o x A T P (300 uM) was applied 2 hr prior to, and for the duration of, peptide application. Representative results are presented in Figure 3-2A. B z A T P (300 uM) evoked a small increase in [Ca ]j (measured by change in wavelength ratio of 340 nm to 380 nm) in untreated microglia (Figure 3-2A, Control, n=28 cells). This increase in [Ca 2 +]j was sustained and remained elevated above basal level at 10 min following application of B z A T P . In microglia pre-treated with Api_42, B z A T P caused a large increase in [Ca 2 +]j which continued to grow in amplitude to a plateau level at 10 min post-application of the purinergic ligand (Figure 3-2A, n=32 cells). However, the B z A T P response was almost totally abolished i f o x A T P was included in the treatment solution with A p M 2 peptide (Figure 3-2A, n=l8 cells). Quantification of BzATP-induced C a 2 + responses with the different treatments was determined by measurement of the peak amplitudes of [Ca 2 +]j. With A P 1 4 2 pretreatment (n=5 experiments; 95 cells), response amplitudes were increased by 145% relative to control (n=4 experiments; 87 cells) (Figure 3-2B). With o x A T P included 119 together with A P 1 - 4 2 in the pretreatment solution (n=4 experiments; 82 cells), Ca' responses were decreased by 86% relative to peptide alone. 120 A B z A T P 3 0 0 | i M pretreatment with A p 1 - 4 2 no pretreatment (control) pretreatment with A p 1 - t t + o x A T P i i i r -1 i i 100 200 300 400 500 600 700 Time (sec) B Amplitudes of responses induced by BzATP control 1-42 A P , _ 4 2 + o x A T P pretreatment Figure 3-2. Changes in [Ca2+]j induced by BzATP A: Application of B z A T P (at 300 uM) increased intracellular C a 2 + (measured by changes in F340/F380). Representative traces show control (no peptide pre-treatment, n=28 cells), A p , 4 2 pretreatment (cells exposed to peptide for 18 hr prior to application of B z A T P , n=32 cells) and peptide pre-treatment in the presence of ox A T P (300 uM) which was added 2 hr prior to peptide (n=18 cells). B: Amplitudes of responses induced by B z A T P for the different treatments. * P < 0.05 compared to control. ** P < 0.05 compared to A p , 4 2 pretreated group. 121 3.3.4 P 2 X 7 R Expression in Rat Hippocampus We next investigated the expression of P 2 X 7 R in rat hippocampus for three different groups of animals; those receiving intra-hippocampal injections of P B S , A(3i_42 or AP42-1. Peptides were injected at lnmol /wi th all analyses carried out at 3 d post-injection. Representative R T - P C R is presented in Figure 3-3A showing a low basal level of P 2 X 7 R following P B S injection. A considerably enhanced expression of the purinergic receptor was observed with injection of APi. 4 2 whereas Ap 4 2 _i was associated with a low level of P 2 X 7 R (Figure 3-3A). Semi-quantitative results for the three groups of injected animals (n=3 for each) are shown in Figure 3-3B. The expression of P 2 X 7 R was increased four-fold in APi. 4 2-injected, relative to P B S or reverse peptide injected, rat hippocampus. We also used immunohistochemical procedures to investigate expression of P 2 X 7 R protein in the dentate gyrus (Figure 3-4A, top, left panel, portion of superior blade denoted by dotted line). Within the superior blade region, minimal P 2 X 7 R immunoreactivity (ir) was evident at 3 days following injection of P B S (Figure 3-4A, top, right panel). However, ir for P 2 X 7 R was markedly elevated in A P 1 . 4 2 , but not A p 4 2 . i , injected animals (Figure 3-4A, bottom panels). Quantification of P 2 X 7 R ir demonstrates the extent of P 2 X 7 R ir induced with A p M 2 injection compared with P B S or A p 4 2 . i injected animals (Figure 3-4B, n=4 for each group). We next ' used double immunostaining to enable identification of the specific cells which were associated with expression of P 2 X 7 R . Representative double immunofluorescent staining is presented in Figure 3-4C for P 2 X 7 R with OX-42 (microglial marker, left panel) and G F A P (astrocyte marker, right panel). The results showed a co-localization of P 2 X 7 R and OX-42 (+)ve 122 microglia within the hippocampus after A p 1.42 injection (Figure 3-4C, left panel). Only a small subset of G F A P (+)ve astrocytes were associated with P 2 X 7 R (Figure 3-4C, right panel). N o co-localization of the purinergic receptor with N e u N (+)ve neurons was observed (data not shown). 123 B u Rat hippocampal tissue j> 2 5 | 2.0 m i l l 1 T X 1.0 • 7 « 0.5 P-actin | „ Q 13 Figure 3-3. In vivo expression and levels of P2X 7 R in rat hippocampus A : R T - P C R for P 2 X ? R in rat hippocampus 3 d post-injection with P B S , A p , 4 2 or A P 4 2 , (peptides at 1 nmol). B: Relative m R N A levels for P 2 X 7 R with different treatments (n=3 for each). * P < 0.05 compared to PBS-injected group. ** P < 0.05 compared to A p M 2 -injected group. * _ 1 ** • • • PBS APL42 AP42-1 124 A P2X 7 R immunoreactivity in rat hippocampus Figure 3-4. Single and double immunostaining for P2X ? R A : A portion of the superior blade is indicated by the dotted region (upper left panel). The other panels of Fig A show expression of P 2 X 7 R in the indicated region at 3 d following injections of PBS (upper right panel), A p , _ 4 2 (lower left panel) and A P 4 2 , (lower right panel); Scale bar = 150 Llm. B: Quantification of P 2 X ? R immunoreactivity for the different treatments (n=4 for each). * P < 0.05 compared to PBS-injected group. ** P < 0.05 compared to A p , 4 2-injected group. C : Representative double staining for P 2 X ? R (green marker) for microglia (red marker, left panel) and astrocytes (red marker, right panel); Scale bars = 30 |Jm. 125 3.4 DISCUSSION A n important and novel finding from this study is that expression of P 2 X 7 R is upregulated in microglia in A D , compared with levels in N D , brain. We also report that human fetal microglia stimulated with APi_4 2 peptide exhibit considerably elevated expressions of P 2 X 7 R relative to control (untreated cells and Ap 4 2 _ i ) and show marked increases in [Ca 2 + ] i in response to a P 2 X 7 R agonist after exposure to peptide. In addition, P 2 X 7 R was markedly upregulated in A P M 2 injected, compared to P B S or Ap 4 2 . i-injected, rat hippocampus. Microglia obtained from N D brain show a low level of expression for P 2 X 7 R (Figure 3-1 A ) . However, a considerable upregulation of the purinergic receptor was evident in A D microglia (Figure 3-1A). Semi-quantitative R T - P C R (Figure 3-1B) indicated an approximate two-fold increase in m R N A levels in A D , relative to N D , microglia. The constitutive expression of P 2 X 7 R in N D microglia could be associated with a relatively low basal activation of cells. Microgl ia in N D brain exhibit a predominantly ramified morphology, however, some ameboid-shaped cells were observed (McLarnon et al., 2005). Cells obtained from A D individuals present a primary ameboid morphology (McLarnon et al., 2005). The respective ramified and ameboid morphologies are commonly associated with resting and activated states of microglia (Walker et al., 1995). The enhanced expression of P 2 X 7 R in A D microglia suggests possible roles for the purinergic receptor in shaping inflammatory activity in A D brain. In this regard, our findings are relevant to the report that P 2 X 7 R is upregulated in a transgenic mouse model (Tg2576) of A D (Parvathenani et al., 2003). The latter study also reported that activation of the purinergic receptor in microglia induced cortical neuronal 126 cell death in a co-culture system indicating the P2X7R may play a role as a contributing factor in neurodegeneration. The stimulation of microglia by A P 1 . 4 2 has been suggested as a suitable in vitro experimental tool for evoking inflammatory responses with relevance to A D brain (Floden et al., 2005; McLarnon et al., 2005). Cultured human microglia exposed to APi_4 2 (5 u M for 18 hr) exhibited a marked increase in the expression of P 2 X 7 R compared with P B S or reverse peptide-treated cells. Relative m R N A levels were increased by 106% in microglia exposed to A P 1 . 4 2 compared with P B S . Together with the results from Figure 3-1, our findings suggest low constitutive expressions of the purinergic receptor in unstimulated microglia. It is possible that increased expression of P2X7R is a step in the activation process of microglia; alternatively high levels of P 2 X 7 R may be manifest subsequent to microglial activation. Functionally, microglia pretreated with APi. 4 2 (5 u M for 18 hr) showed an increased amplitude of [Ca 2 +]j compared to control when stimulated with the P2X7R agonist B z A T P . Inclusion of o x A T P in the pretreatment solution largely blocked the BzATP-induced increase in [Ca 2 +]j. These results would be consistent with a functional role, for P 2 X 7 R in mediating Ca -dependent microglial inflammatory responses under conditions with particular relevance to A D brain. Interestingly, recent work from this laboratory (McLarnon et al., 2005) has reported dysfunctional handling of C a 2 + in A D microglia (compared with N D cells) and in APi. 4 2 treated human microglia (compared with untreated cells). The altered C a 2 + mobilization included smaller amplitudes of A T P -induced responses for both A D microglia and peptide-treated cells. These responses were attributed to release of C a from endoplasmic reticulum (ER) stores subsequent to A T P 127 binding to P 2 Y subtype receptors. A n important point is that these experiments employed A T P at 100 u M , a level insufficient to activate P 2 X 7 R in human microglia (McLarnon, c 2005). Expression of P 2 X 7 R was markedly upregulated in A(3i_42-injected rat hippocampus compared to P B S or reverse peptide-injected brains. Double immunostaining procedures showed microglia were the primary cell type expressing P 2 X 7 R . A small degree of P 2 X 7 R ir was associated with astrocytes whereas neurons exhibited no expression for the purinergic receptor. Although intra-hippocampal injection of AP 1.42 represents a simplified model of A D brain, the in vivo results support roles for P 2 X 7 R in mediating microglial responses to peptide stimulation. The results suggest the utility in testing for effects of pharmacological modulation of P 2 X 7 R in animal models of A D . However, experiments would likely require systematic injection of o x A T P since the compound is not permeable through the blood brain barrier. Overall, our findings indicate the involvement of P 2 X 7 R in mediating inflammatory responses in A D brain. However, a proviso is that even though P 2 X 7 R expression in microglia is enhanced in A D brain or by exposure to A P 1 . 4 2 , elevated A T P concentrations in excess of 1 m M may be required for activation of this receptor (Hide et al., 2000; McLarnon, 2005). Such high levels of A T P would likely be associated with a localized environment of chronic cellular damage and sustained exposure to a brain insult such as amyloid beta peptide. A n important question to be determined in future work is whether animal models of A D show upregulation of P 2 X 7 R with long duration applications of low (nM) levels of APi_42 peptide. 128 3.5 R E F E R E N C E S Burnstock G . 1996. P2 purinoceptors: historical perspective and classification. Ciba Found Symp 198:1-28. Brough D , LeFeuvre R A , Iwakura Y , Rothwell N J . 2002. Purinergic (P2X7) receptor activation of microglia induces cell death via an interleukin-1-independent mechanism. M o l Cel l Neurosci 19:272-80. Choi H B , Hong S H , R y u J K , K i m S U , McLarnon JG. 2003. Differential activation of subtype purinergic receptors modulates C a 2 + mobilization and C O X - 2 in human microglia. G l i a 43(2):95-103. Ferrari D , Chiozzi P, Simonetta F, Hanau S, D i Virgi l io F. 1997. 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Cel l Calcium 27:205-12. Webster S, Bradt B , Rogers J, Cooper N . 1997. Aggregation state-dependent activation of the classical complement pathway by the amyloid beta peptide. J Neurochem 69:388-98. 130 C H A P T E R 4: D I F F E R E N T I A L A C T I V A T I O N O F PURINERGIC R E C E P T O R S M O D U L A T E S L E V E L S O F COX-2 A N D [Ca2+]j IN H U M A N M I C R O G L I A 3 4.1 INTRODUCTION Microglia mediate a diversity of immune responses in the brain including phagocytosis and secretion of a host of agents. In the latter case an important consideration is that microglial responses, such as production of pro-inflammatory cytokines and reactive oxygen species, may lead to damage of neurons (Banati et al., 1993; Colton et al., 1994; Matsuo et al., 1995). Indeed, activated microglia have been implicated in various neurological diseases such as Parkinson's disease (PD) (McGeer et al., 1988; Gao et a l , 2002), multiple sclerosis (MS) (Prineas et a l , 1993; Copelman et al., 2001), Alzheimer's disease (AD) (Tooyama et al., 1990; Lue et al., 2001), and amyotrophic lateral sclerosis (ALS) (Aimer et al., 2001). A T P activates different subtype purinergic receptors in brain with families designated as metabotropic (P2YR) or ionotropic (P2XR) (Ralevic and Burnstock, 1998; Burnstock, 1999). In rodent and human microglia, acute A T P application causes a rapid, • • 2+ transient increase in cytosolic [Ca ]j principally due to release from endoplasmic reticulum (ER) stores (Wang et al., 2000 Moller , 2002). This pathway involves A T P binding with P 2 Y receptors which are G protein coupled to phospholipase C and subsequent production of inositol triphosphate (IP3) which then acts to release C a 2 + from E R stores. In human microglia we have reported that A T P binding to P 2 X subtype receptors leads to activation of non-selective cationic channels which pass influx of N a + and possibly C a 2 + , leading to cell depolarization (Wang et al., 2000). The P2XR-mediated 3 A version of this chapter has been published for publication. Choi HB, Hong SH, Ryu JK, Kim SU, McLarnon JG. (2003) Differential Activation of Purinergic Receptors Modulates Levels of COX-2 and [Ca 2 +]iin Human Microglia. Glia 43(2):95-103. 131 cell depolarization was found to block influx of Ca through store-operated channels (SOC), activated following P 2 Y induced depletion of stores, suggesting interaction between the two subtypes of purinergic receptors (Wang et al., 2000). Upregulation of cyclooxygenase (COX-2) has been suggested as an important contributor to inflammation-induced cytotoxicity (Smith et al., 1996). The expression of C O X - 2 is markedly enhanced in inflamed tissue and is responsible for the production of prostanoid mediators of inflammation including prostaglandins (Seibert et al., 1994; Smith and DeWitt, 1995). Inflammatory stimuli such as lipopolysaccharide (LPS) and cytokines such as T N F - a and I L - i p have been found to enhance C O X - 2 expressions in microglia (Minghetti and Levi , 1995; Bauer et al., 1997). In pathological conditions, elevated C O X - 2 expressions have been observed in A D brain and A L S spinal cord (Ho et al., 1999; Yasojima et al., 1999, 2001). We hypothesized that pharmacological manipulation of purinergic subtype P 2 Y R 2_|_ and P 2 X R could modulate Ca -mediated responses and microglial expression of C O X - 2 . In this study we have used P P A D S as an antagonist for P 2 X R (Denda et al., 2002; Sesti et al., 2002) and A D P - p - S as an agonist for P 2 Y R (Wang et al., 2000) receptors. A n important question to be examined was whether differential activation of P 2 X R and P 2 Y R induced changes in Ca mobilization which could be linked with altered levels of C O X - 2 . 132 4.2 M A T E R I A L S AND M E T H O D S 4.2.1 Preparation and Culture of Human Microglia The methods used in the isolation and identification of microglia have been described previously ( K i m et al., 1986; Satoh and K i m , 1994; Satoh et al., 1995; Nagai et al., 2001). In brief, human embryonic brain tissues (12-18 weeks gestation) were used for the preparation of microglia. Human embryonic brain tissues were dissected into small blocks, incubated in phosphate buffered saline (PBS) containing 0.25% trypsin and 40 p.g/ml DNase for 30 min at 37 °C and dissociated into single cells by repeated pipetting. Dissociated cells were plated in T75 flasks in a medium consisting of Dulbecco's modified Eagle's medium ( D M E M ) with high glucose containing 5% horse serum, 25 ug/ml gentamicin, and 2.5 p.g/ml amphotericin B . Free-floating microglia were harvested from a medium of mixed cell cultures after 7-10 days of growth in culture flasks and plated on aclar coverslips for identification, on poly- L-lysine coated glass coverslips for calcium spectrofluorometry arid plated on 6-well multiplates for R T - P C R . Immunostaining was performed on isolated cells using C D l l b and ricinus communis agglutinin ( R C A ) , specific markers for microglia, and the purity of human microglia was in excess of 98% (Walker et al., 1995; Nagai et al., 2001). The use of embryonic human tissues was approved by the Clinical Screening Committee for Human Subjects of the University of British Columbia. 4.2.2 Calcium Spectrofluorometry The procedures used in Ca 2 +-sensitive fluorescence microscopy in this laboratory have been previously described (Wang et al., 2000; Khoo et al., 2001; McLarnon et al., 133 2001). In brief, the fluorescent Ca indicator fura-2 acetoxymethylester (Fura-2/AM) was added with pluronic acid (both agents at 1 uM) to human microglia bathed in physiological saline solution (PSS) at room temperature (20-22 °C) for 30 min. Cells were washed for 10 min in dye-free PSS. Coverslips were placed on the stage of an inverted microscope (Zeiss Axiovert with 40x quartz objective) and alternating 340 nm and 380 nm U V light was used for excitation. Fluorescent signals were recorded by passing 510 nm of emission light (bandwidth of 40 nm) to a digital camera ( D V C 1310, D V C Co. , Austin, T X ) . A n imaging system (Empix, Mississauga, O N , Canada) was used to process and analyze fluorescent signals based on the wavelength ratios (340/380 nm) and ratio images were processed every 8 s. Values for ratios are reported with no conversion to absolute levels of C a 2 + ; however, a change in ratio of 0.1 would correspond to a change in intracellular C a 2 + [Ca 2 +]j of about 120 n M . A l l imaging experiments were done at room temperature. 4.2.3 Reverse-Transcription P C R (RT-PCR) R T - P C R was used to detect m R N A from human microglia stimulated with purinergic agonists and antagonists. Cultured cells were treated with the following agents: A T P (100 uM) , P P A D S (10 uM), a selective P 2 X R antagonist or A D P - 0 - S (100 uM) , a selective P 2 Y R agonist either separately or together for up to 8 hr. P P A D S (10 uM) was applied 20 min ahead of A T P application. Total R N A was isolated by TRIzol ( G I B C O - B R L , Gaithersburg, M D ) and complementary D N A ( c D N A ) synthesis was carried out using M M L V reverse-transcriptase ( G I B C O - B R L ) . Human specific C O X - 2 signals were generated with the Gene E thermal cycler (Techne, NJ) and Amplitaq Gold 134 D N A polymerase (Applied Biosystems, Foster City, C A ) . Primers used for C O X - 2 were as follows: Sense primer 5' - T T C A A A T G A G A T T G T G G G A A A A T T G C T - 3 ' and antisense primer 5' - A G A T C A T C T C T G C C T G A G T A T C T T - 3 " . P C R product size was 305 bp for C O X - 2 . Conditions for P C R were as follows: initial denaturation 95 °C for 8 min followed by 35 cycles of denaturation at 95 °C for 1 min, annealing at 60 °C for 1 min, extension at 72 °C for 2 min. Glyceraldehyde-3-phosphate dehydrogenase ( G A P D H ) was used as a reaction standard (Ercolani et al., 1988). P C R products were identified using 1.5% agarose gels containing ethidium bromide and visualized under U V light. 4.2.4 Immunocytochemistry Human microglia cells grown on poly-L-lysine coated Aclar plastic coverslips were incubated in D M E M for 24 hr and then treated with A T P , A T P with P P A D S , P P A D S or ADP-P-S. After 12 hr incubation, cells were fixed in 4% paraformaldehyde in 0 .1M Phosphate buffer for 10 min, washed with P B S , and then permeabilized in 0.2% Triton X-100 in 0 .1M P B S for 20 min. Cells were incubated in rabbit ant i-COX-2 (1:200 dilution; Chemicon, Temecula, C A ) at 4°C for 48 hr and followed by Alexa Fluor 488 anti-rabbit IgG secondary antibody (1:100; Molecular Probes, Eugene, OR) incubation at R T for 1 hr in the dark. After P B S washes, coverslips were then immersed in 4',6-diamidino-2-phenylindole (DAPI) (Molecular Probes) at 1 (.ig/ml in P B S to visualize cell nuclei and to determine total cell numbers. The coverslips were mounted onto glass slides using gelvatol and examined under a Zeiss fluorescence microscope and photographed using a cooled C C D camera. Stained cells were counted in at least four random fields and 135 the ratio of C O X - 2 positive cells to total cell number per field at X200 magnification was calculated. 4.2.5 Cellular Morphology and Viability The effects of purinergic stimulation on morphology of cultured human microglia was assessed in this study. Microgl ia were first incubated in serum-free medium for 48 hr which yielded a homogenous population of cells with a ramified, process bearing, morphology. A 20 hr exposure of microglia to purinergic agents was then carried out and the number of cells showing an ameboid morphology was then estimated. The criteria for I identification of cells with an ameboid shape was a roundish profile with no processes. D A P I staining was routinely used to examine the viability of microglia with different treatments. Cells with round and intact nuclei were considered indicative of healthy cells. We found no evidence for nuclear condensation and fragmentation under any of the experimental conditions indicating no loss of cell viability in the present experiments. 4.2.6 Solutions and Reagents The spectrofluorometry studies employed a standard PSS solution containing (in *rnM): N a C l (126), KC1 (5), C a C l 2 (1), MgCl 2 (1 .2 ) , H E P E S (10), D-glucose (10); p H 7.4. In some experiments a Ca 2 +-free PSS was used, this solution had the same ionic composition as standard PSS with the exception that it included EGTA.(a t 1 m M ) with no added C a C l 2 . The fluorescence indicator fura-2/AM was purchased from Molecular \ 136 Probes (Eugene, OR). A n d all other agents used in the present study including A T P , A D P - p - S , P P A D S and SKF96365 were obtained from Sigma (St. Louis, M O ) . 137 4.3 R E S U L T S 4.3.1 Characterization of [Ca ]j Responses by Purinergic Receptor Modulators Transient increases in [Ca ]j induced by A T P (atTOO u M or below) are largely mediated by E R stores release in rodent (Moller et al., 2000) and human (McLarnon et al., 1999) microglia. These responses are initiated by A T P binding to the P 2 Y subtype of purinergic receptors. A representative experiment is shown in Figure 4-1A and the analysis of the response used a measure of half-maximum time T 1/2 (time at one-half of peak amplitude). In this case the A T P response had a T1/2 of 22 s. Overall, the mean value of T1/2 for A T P induced increases in [Ca 2 + ]i was 26.7 + 0.5 s (n = 266). When A T P was applied in the presence of P P A D S , a prolonged time course of [Ca Z T] i was evident (Figure 4-1B); in this experiment a T i / 2 of 90 s was measured. Overall, the mean time was 82.0 ± 2.6 s (n = 116) a significant increase relative to that measured with A T P alone. A s 94-expected P P A D S alone, in the absence of A T P , had no effect to alter [Ca ]i (inset of Figure 4-1B) a result found with a total" of n = 64 cells. As-shown in Figure 4-1C, the 94-P 2 Y R agonist ADP-f i -S (applied at 100 uM) also elicited a lengthened [Ca ]j response compared with that using A T P (Figure 4-1 A ) ; in this experiment a T1/2 o f 96 s was measured. Over the total number of cells which were studied, the mean value of half-time for the increase in [Ca 2 +]j induced by A D P - p - S was 106.4 + 3.6 s (n = 97). Thus, reducing the effects of P 2 X R mediated contributions leads to a tripling (with the P 2 X R antagonist P P A D S ) or quadrupling (with the P 2 Y R selective agonist ADP-P-S) of T i / 2 relative to that measured with A T P as a ligand. 2+ 2+ The introduction of Ca -free PSS reduced [Ca ]; to baseline levels suggesting that the sustained component of A T P responses, evident in Figure 4 - IB with P 2 X R 138 inhibition or in Figure 4-1C with P 2 Y R activation, was due to influx of Ca . This point was systematically examined in the following set of experiments using A T P plus P P A D S to stimulate cells. Initially, Ca 2 +-free solution was applied immediately following attainment of a plateau phase of [Ca ], induced by A T P and in the maintained presence of P P A D S (Figure 4-2A). In this, and 3 other experiments (n = 196), application of C a 2 + -free PSS totally abolished responses. Store-operated channels (SOC) constitute a primary influx pathway in human (Wang et a l , 2000) and rodent (Toescu et al., 1998) microglia. To investigate i f S O C mediated the sustained component of [Ca 2 +]j, the S O C inhibitor SKF96365 was applied following attainment of the plateau phase of [Ca 2 +]j. A s shown in Figure 4-2B, SKF96365 (at 50 uM) caused a rapid inhibition of [Ca 2 +]j to basal level; application of Ca -free PSS had no additional effect to reduce the response. This result was obtained in a total of n = 172 cells. In summary, application of either Ca 2 +-free PSS or PSS containing SKF96365 abolished the prolonged phase of the A T P response induced in the presence of a P 2 X R inhibitor. We next examined the possible involvement of the P 2 X 7 (formerly termed P2Z) subtype purinergic receptor in mediating changes in [Ca 2 +]j. In these experiments the P 2 X 7 R ligand benzoylbenzoyl A T P (BzATP) was applied at a concentration of 100 u M . A representative response is presented in Figure 4-2C showing B z A T P had no effect to alter [Ca 2 +]j. Overall, in n = 50 cells, application of this selective P 2 X 7 R agonist elicited no calcium response from cultured human microglia. 139 Figure 4-1. Modulation of ATP induced changes in [Ca ]j A: A T P (100 u M ) elicited a rapid transient change in [Ca ]j (mean response, n = 23 cells). B: The P 2 X R antagonist P P A D S (10 uM) was applied prior to eliciting an A T P response (mean response, n = 19 cells). C: Application of the P 2 Y R selective agonist ADP-P-S (at 100 u M ; mean response, n = 15 cells). 140 Ca free PSS B 0.7 0.6 0.5 o r^> 0.4 I 0.3 m to 0.2 0.1 0.0 0.7 0.6 0.5 o - 0 . 4 0.2 0.1 0.0 150 200 250 Time (sec) PSS P P A D S 300 350 400 Ca- 2 + free PSS A T P PSS PPADS 50 100 150 200 250 300 350 400 50 100 150 200 250 300 350 400 Time (sec) PSS ADP-(3-s C a 2 + free PSS 50 100 150 200 250 300 350 400 Time (sec) 141 Figure 4-2. [Ca z ]j responses elicited by A T P plus PPADS and with BzATP A: A T P + P P A D S was used to induce a plateau phase of a [Ca 2 +]j response prior to addition of Ca 2 +-free PSS (mean response, n = 34 cells). B: A similar procedure as in (A) where SKF96395 (at 50 uM) , was applied in standard C a 2 + P S S , following attainment of the plateau phase of a [Ca 2 +]j response (mean response, n = 20 cells). C: Application of the P 2 X 7 R agonist B z A T P (at 100 uM) (mean response, n = 18 cells). 142 0.7 0.6 0.5 0.4 PSS Ca 2 + free PSS o I 0.3 0.2 0.1 0.0 P P A D S A T P 50 100 150 200 250 300 350 400 450 Time (sec) PSS Ca 2 + free PSS — i 1 1 1 1 1 1 1 1 50 100 150 200 250 300 350 400 450 Time (sec). 0.7 0.6 o fl5 oo m 0.4 3 0.3 0.2 0.1 0.0 PSS B z A T P 50 100 Time (sec) 150 200 143 4.3.2 Effects of Purinergic Receptor Modulators on COX-2 mRNA Expression We then used R T - P C R to investigate the relationship between activation of P 2 X R and P 2 Y R and expression of C O X - 2 in human microglia. A secondary question examined was the possible role of [Ca ]i in linking purinergic receptors with the enzyme. Under basal unstimulated conditions, the level of C O X - 2 m R N A was undetectable in human microglia (Figure 4-3A, lane 1). Treatment with A T P (100 u M for 8 hr) had no effect to induce expression of C O X - 2 m R N A (Figure 4-3A, lane 2). However, when microglia were exposed to A T P plus P P A D S , a significant increase in C O X - 2 expression was found (Figure 4-3A, lane 3). A similar result was obtained for treatment with ADP-P-S, whereby expression of C O X - 2 was significantly enhanced (Figure 4-3 A , lane 4). Similar results were obtained in three additional R T - P C R studies; that is C O X - 2 was not constitutively expressed or present with A T P treatment but was expressed with inhibition of P 2 X R ( A T P plus P P A D S ) or stimulation with a P 2 Y R agonist. A second R T - P C R study was designed to test for C a 2 + dependence in the purinergic induction of C O X - 2 expression. Specifically, a role for SOC was examined in 7-1-two ways; by using treatment in Ca -free medium or treatment with SKF96365 added to standard C a containing medium. In both cases P P A D S was added with A T P for the 8 hr treatments. Representative R T - P C R for one set of experiments are shown in Figure 4-3C. A s expected, A T P alone induced no C O X - 2 (Figure 4-3C, lane 2). However, microglia treated with A T P plus P P A D S caused expression of C O X - 2 (Figure 4-3C, lane 3) which was abolished in Ca -free medium where A T P plus P P A D S had no effect to induce C O X - 2 m R N A (Figure 4-3C, lane 4). Treatment using SKF96365 (at 25 uM) added to 144 A T P plus P P A D S in standard C a containing medium showed no expression of C O X - 2 (Figure 4-3C, lane 5). P P A D S alone had no effect, in this and a second study, to induce C O X - 2 m R N A in human microglia (Figure 4-3C, lane 6). 145 A bp M 1 2 3 4 400 300 COX-2 B 400 300 , , M mm G A P D H COX-2 G A P D H Figure 4-3. R T - P C R for purinergic modulation of COX-2 expression in treated human microglia A : Expression o f C O X - 2 for unstimulated control (lane 1); for A T P (lane 2); for A T P + P P A D S (lane 3); for ADP-P-S (lane 4). Four independent experiments showed similar results. B: Glyceraldehyde-3-phosphate dehydrogenase ( G A P D H ) was used as a reaction standard. C: Expression of C O X - 2 (different R T - P C R study from above) for unstimulated control (lane 1); for A T P (lane 2), for A T P + P P A D S (lane 3), for A T P + P P A D S in Ca 2 +-free PSS (lane 4); for A T P + P P A D S with added SKF96365 (lane 5) and for P P A D S alone (lane 6). Two independent experiments showed similar results. D: G A P D H standard. 146 4.3.3 P2XR Modulate COX-2 Protein Expression The effects of pharmacological modulation of purinergic receptors on the production of C O X - 2 were studied using an antibody specific for the enzyme. In control (unstimulated cells) there was no evidence, for C O X - 2 -positive cells (Figure 4-4A). Overall, only 2.8 ± 0.7% of the total number of cells in control stained positive for C O X -2 (Figure 4-4F). Following exposure of microglia to A T P (100 u M for 12 hr), the number of C O X - 2 -positive cells was increased compared with untreated controls ( A T P , 23.6 ± 3%; p < 0.05; Figure 4-4C and F). We then investigated the effect of P 2 X R modulation on C Q X - 2 protein expression using the same treatment paradigm as noted above. A s shown in Figure 4-4D, exposure of microglia to A T P plus P P A D S led to a marked enhancement in C O X - 2 production relative to A T P alone ( A T P plus P P A D S , 55.7 ± 5%; p < 0.05). A similar result was found for treatment with ADP-P-S (Figure 4-4E) where C O X - 2 levels were significantly higher compared with A T P (ADP-p-S , 39.4 ± 5%; P < 0.05; Figure 4-4F). P P A D S alone (Figure 4-4B, F) had little effect to increase protein expressions ( P P A D S , 6.2 ± 0.8%>). These results show that not only expressions of C O X - 2 , but also protein levels of the enzyme, are altered with modulation of A T P receptors. 147 Figure 4-4. Purinergic modulation of COX-2 production in human microglia Cells were treated for 12 hr in solutions and were immunostained with a C O X - 2 antibody prior to quantitation. A : Unstimulated control; B: P P A D S alone; C: A T P ; D: A T P plus P P A D S ; E : A D P - p - S ; F: A bar graph showing the percentage of C O X - 2 positive microglia for the different treatments (expressed as % of total). Data represent the mean ± S E M . Four independent experiments were carried out for each condition. * p < 0.05 compared with control # p < 0.05 compared with A T P . A N O V A and Student-Newman-Keuls. 148 A B 149 4.3.4 Effects of Purinergic Agents on Morphology of Microglia Microgl ia generally exhibit two distinct types of morphology categorized as ramified and ameboid and which, in a simplified designation, may represent inactivated and activated states, respectively (Kreutzberg, 1996; Nakamura et al., 1999). During the course of the R T - P C R studies described above, it was apparent that pharmacological manipulation of purinergic receptors with chronic A T P treatment had effects to alter morphology of cultured human microglia. This observation was systematically examined by first incubating cells in serum-free medium for 48 hrs. This procedure yields a relatively homogenous population of microglia with a ramified type of morphology. Following incubation in serum-free medium several different protocols were then applied which consisted of unstimulated (control) and 20 hr treatments of cells with A T P alone (100 uM) , A T P plus P P A D S , P P A D S alone and A D P - p - S . The results are presented in Figure 4-5 and show that for control (Figure 4-5A), and with treatments using A T P (Figure 4-5B) or P P A D S (Figure 4-5D), the morphology of microglia was predominantly ramified (estimated to be in excess of 90% of total cells). However, for microglia treated with either A T P plus P P A D S (Figure 4-5C) or with ADP-P-S (Figure 4-5E), cells showed a shift to an ameboid morphology (ameboid shapes represented more than 50% of cells). For comparison purposes the inflammatory stimulus L P S , a potent activator of microglia, was also applied to cells. A s shown in Figure 4-5F, L P S (100 ng/ml) induced an ameboid morphology for close to 100%) of treated cells. Thus, although the cells with ameboid shapes can only be considered qualitatively, differential activation of P 2 X R and P 2 Y R appears to modulate the morphology, and possibly the degree of activation, of human microglia. 150 Figure 4-5. Morphological changes in human microglia following treatments Cultured human microglia were initially incubated in serum-free medium for 48 firs. Representative phase contrast morphologies following 20 hr treatments for A : unstimulated control, B: A T P , C: A T P + P P A D S , D: P P A D S , E : A D P - p - S and F: L P S . The latter was used for comparison purposes as a potent activator of human microglia. 151 4.4 DISCUSSION This study has documented effects of the differential activation of purinergic P 2 Y R and P 2 X R on Ca mobilization and cellular expression and production of C O X - 2 in human microglia. The results suggest that influx of C a 2 + through SOC plays a role in linking A T P activation of the different subtypes of purinergic receptors with cell function. A specific, novel finding was that reducing the contribution of the P 2 X R mediated pathway, with either a competitive inhibitor of this receptor or using a P 2 Y R selective agonist, enhanced C O X - 2 expression in human microglia. This result has implications in the pharmacological modulation of microglial responses in brain inflammation. The primary contribution to ATP-induced transient increases in [Ca 2 + ] , arises from depletion of E R stores a process coupled to P 2 Y subtype receptors (McLarnon et al., 1999; Moller et al., 2000; Wang et al., 2000). In this work we found marked enhancements in time courses of [Ca 2 +]j with application of A T P plus P P A D S or ADP-P-S relative to A T P applied alone (Figure 4 - l B , C ) . This result reflects prolongation of a slower, sustained phase of the purinergic response. This secondary component represents entry of C a 2 + since it was abolished with Ca 2 +-free PSS (Figure 4-2A). The influx J pathway is consistent with SOC mediated entry since SKF96365 completely blocked the maintained response (Figure 4-2B). Thus, our results suggest that A T P activation of the ionotropic P 2 X subtype purinergic receptor normally inhibits S O C mediated influx. A possible mechanism is that influx of N a + through the non-selective cationic channel coupled to P 2 X R causes cell depolarization and reduction of the driving force for C a 2 + influx mediated by SOC. 152 We tested i f the P2X7 subtype of purinergic receptor may be involved by using application of the agonist benzoylbenzoyl A T P (BzATP) (applied at 100 uM). A t this concentration there was no effect of B z A T P to alter [Ca 2 +]j (Figure 4-2C) suggesting little or no contribution from P 2 X 7 R in modulating P 2 Y R dependent responses. This result is consistent with previous findings showing that activation of P 2 X 7 R in microglia often requires higher concentrations of A T P at levels at or above 1 m M (Ferrari et al., 1997). A t present, the specific subtype of purinergic P 2 X R which may mediate depolarization and inhibit entry of Ca through S O C in human microglia is not known. Chronic treatment of human microglia showed differential activation of subtypes of purinergic receptors altered cellular function. The use of either P P A D S added with A T P or the selective P 2 Y R agonist ADP-P-S produced equivalent results in that expression ( R T - P C R assay, Figure 4-3) and production (immunocytochemistry, Figure 4-4) of inducible C O X - 2 were significantly increased relative to that measured with A T P . Indeed, no expression of C O X - 2 was observed either constitutively or following application of A T P which would activate both P 2 X R and P 2 Y R (Figure 4-3). However, the more sensitive immunocytochemistry assay did show A T P to increase production of C O X - 2 compared with untreated cells (Figure 4-4F). Thus, activation of P 2 X R by A T P normally inhibits, but does not abolish, production of C O X - 2 . The parallel findings for effects of modulation of purinergic receptors on [Ca 2 + ]i responses and expressions/productions of C O X - 2 indicates that changes' in C a 2 + mobilization are correlated with altered cell function. Specifically, influx of C a 2 + through S O C is coupled to enhancement of C O X - 2 . The increase in C O X - 2 observed with A T P relative to untreated control (Figure 4-4F) may reflect a small contribution of C a 2 + entry 153 / \ to the overall A T P response in the absence of an inhibitor of P 2 X R . This explanation is consistent with studies on human microglia which show A T P responses in Ca 2 +-free solution, relative to standard PSS, are reduced in time course (McLarnon et al., 1999; Wang et al., 2000). Thus in human microglia A T P induced changes in [Ca 2 +]j normally contain a small, but finite, S O C component which could play an important role in cellular functional responses. 2+ The link between C a influx and C O X - 2 likely involves stimulation of phospholipase A 2 ( P L A 2 ) and subsequent downstream effectors. Recent work using a murine microglial cell line has shown dependence of cellular activation on a P L A 2 cascade involving mitogen activated protein kinase and increased arachidonic acid available for C O X - 2 (Paris et al., 2000). This study also demonstrated L P S activation of P L A 2 and that inhibition of C O X - 2 blocked L P S evoked activation of cells. Interestingly, inhibition of P L A 2 and blockade of cyclooxygenase has also been shown to induce reactivity in microglia, but not astrocytes, in spreading depression induced in rat brain (Caggiano and Kraig, 1996). A secondary finding from this work was that differential activation of purinergic receptors appeared to modify the morphology of microglia. It was necessary to first incubate microglia in serum-free medium to produce a homogenous population of cells with ramified morphology (Figure 4-5A). A T P treatment had no effect to alter this morphology (Figure 4-5B). However, treatment with A T P plus P P A D S (Figure 4-5C) or ADP-P-S (Figure 4-5E) had effects to increase the number of cells with ameboid shapes. The shift in profile of morphology may suggest a link between localized entry of C a 2 + through S O C and cellular morphology. If an ameboid configuration is taken to reflect a 154 more activated phenotype then pharmacological manipulation of subtypes of purinergic receptors could modify the degree of cellular activation. Interestingly, a recent study has indicated that long term application of A T P (600 u M for 3 days) led to the ramification of rat microglia (Wollmer et al., 2001). 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Toescu E C , Moller T, Kettenmann H , Verkhratsky A . 1998. Long-term activation of capacitative C a 2 + entry in mouse microglial cells. Neuroscience 86(3):925-35 Tooyama I, Kimura H , Akiyama H , McGeer P L . 1990. Reactive microglia express class I and class II major histocompatibility antigens in Alzheimer disease. Brain Res 523:273-80. Walker D G , K i m S U , McGeer P. 1995. Complement and cytokine gene expression in cultured microglia derived from postmortem human brains. J Neurosci Res 40:478-93. 158 Wang X , K i m S U , van Breeman C, McLarnon JG. 2000. Activation of purinergic P 2 X receptors inhibits P2YR-mediated C a 2 + influx in human microglia. Cel l Calcium 27:205-12. Wollmer M A , Lucius R, Wilms H , Held-Feindt J, Sievers J, Mentlein R. 2001. A T P and adenosine induce ramification of microglia in vitro. J Neuroimmunol 115:19-27. Yasojima K , Schwab C, McGeer E G , McGeer P L . 1999. Distribution of cyclooxygenase-1 and cyclooxygenase-2 m R N A s and proteins in human brain and peripheral organs. Brain Res 830:226-36. Yasojima K , Tourtellotte W W , McGeer E G , McGeer P L . 2001. Marked increase in cyclooxygenase-2 in A L S spinal cord: implications for therapy. Neurology 57(6):952-6. 159 C H A P T E R 5: CONCLUSIONS O F THESIS R E S E A R C H A N D F U T U R E DIRECTIONS This thesis has examined activation of purinergic receptors in microglia. I have measured in vitro and in vivo effects of purinergic stimulation on intracellular signaling pathways and functional responses of microglia using calcium spectrofluorometry, R T -P C R , immunocytochemistry and immunohistochemistry methods. A basic hypothesis of my work is that activation of P 2 X 7 R in microglia is a critical factor in mediating chronic brain inflammation and modulation of P2X 7R-dependent C a 2 + signaling pathways alters expression and production of pro-inflammatory mediators in human microglia. Pharmacological inhibition of P 2 X 7 R and other purinergic receptors has been investigated in detail as a potential therapeutic intervention in decreasing inflammatory responses in microglia in vitro and in vivo. Firstly, I have investigated roles of the purinergic ionotropic receptor, P 2 X 7 R , in mediating lipopolysaccharide (LPS)-induced inflammatory responses and neuronal damage in rat striatum. L P S injection into striatum markedly increased the expression ( m R N A ) and induction (protein) of P 2 X 7 R in microglia. LPS also upregulated a host of pro-inflammatory mediators including inducible nitric oxide synthase ( iNOS, marker for nitric oxide production), 3-nitrotyrosine (3-NT, marker for nitration .of proteins), 4-hydroxynonenal (4 -HNE, marker for l ipid peroxidation) and 8-hydroxy-2'-deoxy guano sine (8-OHdG, marker for oxidative D N A damage). Most importantly, neuronal viability was reduced in LPS-injected brain which suggested as a consequence of inflammatory responses. The P 2 X 7 R antagonist, o x A T P was effective in reducing expression of all inflammatory mediators and conferring protection to striatal neurons. In terms of neuroprotection o x A T P blockade of P 2 X 7 R was found to reduce numbers of 160 caspase-3 (+)ve neurons and increase N e u N (+)ve cell survival in LPS-injected brain. Additionally, o x A T P inhibited LPS-induced activation of the intracellular signaling factor, p38 mitogen-activated protein kinase (p38 M A P K ) and the transcriptional factor, nuclear factor kappa B ( N F K B ) . These results suggest that inhibition of microglial P 2 X 7 R attenuates LPS-induced inflammatory responses leading to neuronal protection. In vitro, R T - P C R assay showed L P S stimulation of cultured human microglia caused an increase in cell expression of a broad spectrum of pro-inflammatory mediators including cyclooxygenase-2 (COX-2) , interleukin-1 P ( IL- ip ) , IL-6, IL-12 and tumor necrosis factor-a (TNF-a) . In the presence of o x A T P , expressions of all inflammatory mediators were significantly reduced. A n interesting finding in this work was that the anti-inflammatory cytokines IL-10 and tumor growth factor-pi (TGF-P 1) were not altered by o x A T P antagonism of P 2 X 7 R signaling. Calcium-sensitive spectrofluorometry showed the P 2 X 7 R agonist B z A T P to cause a sustained increase of [Ca 2 +]j . Pretreatment with L P S was found to markedly potentiate the P2X 7 R-mediated C a 2 + influx in human microglia induced by B z A T P . O x A T P was effective in blocking entry of C a 2 + indicating P2X 7R-mediated increases of [Ca ]j contribute -to microglial activation. These results suggest inhibition of P 2 X 7 R as an anti-inflammatory strategy and novel putative therapeutic approach in the.reduction of microglial activation leading to neuroprotection in inflamed brains. A second experiment examined expression patterns of P 2 X 7 R in microglia from Alzheimer's disease (AD) patients and non-demented (ND) individuals using R T - P C R . Semi-quantitative R T - P C R results showed enhanced expression of P 2 X 7 R in A D microglia compared with N D cells from the analysis of six A D and seven N D cases. 161 These results suggest a significantly enhanced expression of P 2 X 7 R in microglia from A D patients relative to N D individuals. Since A D brain includes deposits of Ap peptide, I then carried out additional experiments to test P 2 X 7 R expression in human fetal microglia stimulated by peptide. R T - P C R results showed amyloid beta (Api_42) stimulated microglia to exhibit a robust expression, for P 2 X 7 R whereas P B S , or reverse peptide (Ap4 2.i) ; treated cells exhibited a low level of expression for this subtype purinergic receptor. A s part of this work, I also examined functional responses mediated by P2X7R 2+ dependent [Ca ]j mobilization in cultured human fetal microglia in the absence and presence of APi. 4 2 stimulation. The selective P 2 X 7 R ligand B z A T P was used to induce [Ca 2 +]j. The amplitudes of C a 2 + responses evoked by B z A T P were increased with APi_ 4 2 pretreatment of cells relative to control (no peptide pretreatment) and were largely blocked i f the P 2 X 7 R inhibitor o x A T P was added with peptide in pretreatment solutions. These results suggest that increased P 2 X 7 R expression is linked to augmented C a 2 + -dependent functional responses in human microglia exposed to APi_42. To complete this component of study, I examined the expression of the P 2 X 7 R in APi_42-injected rat hippocampus; control animals were injected with P B S or the reverse peptide (Ap42„i). Intrahippocampal injections of P B S or Ap 4 2 _i were associated with a low levels of P 2 X 7 R expression whereas injection of APi_4 2 considerably enhanced P 2 X 7 R expression. Semi-quantitative R T - P C R showed that the expression of P 2 X 7 R was significantly increased in APi. 4 2 -injected, relative to P B S or Ap42_i-injected, rat hippocampus. I also investigated expression of P 2 X 7 R protein in the dentate gyrus after intrahippocampal injections of P B S , A p M 2 or Ap 4 2 - i using immunohistochemical 162 procedures. Min imal P 2 X 7 R immunoreactivity (ir) was observed in P B S and A p 4 2 . i -injected groups. However, a markedly elevated P 2 X 7 R ir was evident in APi_42-injected animals. Double immunostaining results showed OX-42 (+)ve microglia were highly co-localized with P 2 X 7 R in APi_42-injected rat hippocampus. The overall results suggest that P2X 7 R-mediated microglial inflammatory responses might play a critical role in neurodegeneration in A D brain. The findings from the first and second components of this work are linked. In vivo the activation of P 2 X 7 R in LPS-injected rat striatum leads to enhancement of a host of cellular inflammatory responses and upregulation of inflammatory mediators including microgliosis, levels of i N O S , oxidative stress, intracellular signaling factors p38 M A P K and N F K B . Importantly, neuronal viability was diminished. The second component of my work showed microglia from A D patients and cultured microglia exposed to APi_4 2had significantly elevated levels of P 2 X 7 R m R N A compared with microglia from N D individuals and unstimulated cells, respectively. Furthermore, intrahippocampal injection of APi_42 markedly elevated expression ( m R N A and protein) of P 2 X 7 R . Thus, conditions relevant to A D brain including use of A D microglia and use of APi-42 peptide as a microglial stimulus induce an inflammatory microenvironment of altered P 2 X 7 R similar to that with direct in vivo injection of L P S the most potent inflammatory stimulus. Although L P S and'Ap i-42 show some similarity in action, it is unlikely the stimuli share the same receptor. L P S shows predominant binding to C D 14 and T L R whereas Ap binds to a variety of putative receptors including the receptor of advanced glycation end products ( R A G E ) , scavenger receptors (SR) and formyl peptide receptor (FPR). 163 A third component of my work involved in vitro study using A T P (100 uM) to stimulate human microglia. Measurements using Ca 2 +-sensitive spectrofluorometry showed A T P (100 uM) to cause rapid, transient increases in [Ca ]j. Application of A T P plus the P 2 X R antagonist, P P A D S or treatment with ADP-P-S, a selective P 2 Y R agonist, led to a considerable prolongation in [Ca 2 +]j responses compared with A T P . The prolonged time courses were consistent with sustained activation of store-operated channels (SOC) since SKF96365, an inhibitor of SOC, inhibited this component of the response. R T - P C R data showed microglia expressed no C O X - 2 m R N A either constitutively or following treatment with A T P (100 u M for 8 hr). However, treatment using A T P plus P P A D S or ADP-p -S alone led to marked expression of C O X - 2 . The enhanced C O X - 2 expression with A T P plus P P A D S stimulation was absent in the presence of SKF96365 or using Ca 2 +-free solution. Immunocytochemistry, using a specific anti-COX-2 antibody, revealed a pattern of purinergic modulation, whereby lack of P 2 X R activation enhanced the production of C O X - 2 protein. These results suggest that modulation of subtypes of purinergic receptors regulates C O X - 2 in human microglia with a link involving S O C mediated influx of C a 2 + . The results summarized above demonstrate that modulation of purinergic 2"T" receptors altered [Ca ]i signaling pathways as well as the expression and production of a pro-inflammatory mediator, C O X - 2 in human microglia. The significance of these results is that modulation of microglial purinergic receptors could serve as a therapeutic strategy in inflammatory conditions of the brain. 164 Even though the results from this work indicate intracerebroventricular injection of o x A T P was effective in blocking P2XvR-mediated microglial activation leading to neuronal protection, it should be noted that o x A T P does not cross the blood-brain barrier ( B B B ) . The B B B is a continuous layer of endothelial cells that regulate the passage of molecules from the circulation into the brain parenchyma. Therefore, the development of methods to allow o x A T P to penetrate the B B B is necessary for the clinical application of this compound. Future research should be carried out to determine whether inhibition of P2X7R in microglia is effective in ameliorating cognitive decline and memory loss evident in A D using a transgenic Tg2576 mouse model of A D . It would be of interest to determine the expression patterns of P2X7R and effects of inhibition of this subtype of purinergic receptor on cognitive decline and memory loss using Tg2576 mice. The role of P2X7R in inflammatory conditions could be further investigated in microglia using P2X7R _ /" knockout mice. These studies w i l l give us better understanding of roles of microglial P2X7R in inflammatory conditions of the brain. 165 

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