Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Beta-Amyloid₁₋₄₂- induced intracellular signaling pathways, functional responses and modulation by 4-aminopyridine.. 2005

You don't seem to have a PDF reader installed, try download the pdf

Item Metadata

Download

Media
ubc_2005-103949.pdf
ubc_2005-103949.pdf [ 15.41MB ]
Metadata
JSON: 1.0092879.json
JSON-LD: 1.0092879+ld.json
RDF/XML (Pretty): 1.0092879.xml
RDF/JSON: 1.0092879+rdf.json
Turtle: 1.0092879+rdf-turtle.txt
N-Triples: 1.0092879+rdf-ntriples.txt
Citation
1.0092879.ris

Full Text

Beta-Amyloidi_42-Induced Intracellular Signaling Pathways, Functional Responses and Modulation by 4-Aminopyridine in Microglia by Sonia Franciosi B.Sc , The University of British Columbia, 1998 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES Pharmacology & Therapeutics UNIVERSITY OF BRITISH C O L U M B I A April 2005 © Sonia Franciosi, 2005 A B S T R A C T Alzheimer's Disease (AD) is a progressive, neurodegenerative disease characterized by gradual cognitive decline and memory loss. Although research has focused on elucidating the risk factors, pathophysiologic abnormalities associated with A D and on mechanisms of impeding disease progression, results indicate that a variety of factors may contribute to A D which makes treating this disease difficult. The neuropathological hallmarks of A D include senile plaques which are composed of extracellular deposits of amyloid beta (AP) peptide as well as neurofibrillary tangles, neuronal loss and inflammation. Microglia, the immune cells of the CNS, are abundantly found in the vicinity of neuritic plaques. It is believed that microglia become activated in response to Ap leading to an inflammatory response and subsequent neuronal loss associated with A D pathogenesis. Modulation of the Ap-induced intracellular signaling and functional responses of microglia could serve as a therapeutic strategy for A D . Full length amyloid beta, AP1-42, induced distinct intracellular signaling pathways in human microglia. Electophysiological studies indicated that Api.42 acutely applied to human microglia upregulated the expression of a novel outward K7 current, sensitive to the non- selective K + channel blocker 4-aminopyridine (4-AP). A similar outward K + current was activated by intracellular application of GTPyS which suggests that AP1.42 induces an outward K + current in microglia via a G protein. Molecular biology studies indicated that the K + channel upregulated by A p ^ 2 was likely due to Kv3.1. APi_42 also caused a transient depolarization of microglia and increased the expression of the Fcyll receptor. The Fcyll receptor mediated this depolarization since antibody inhibition of the Fcyll receptor inhibited the APi-42-induced depolarization. In addition to its ability to block the outward K + current upregulated by APi_42, several in vitro and in vivo assays indicated that 4-AP modulates APi-42-induced intracellular signaling and functional responses of microglia including neurotoxicity. Calcium spectrofluorometric studies indicated that AP1.42 activated a calcium entry pathway which was blocked by 4-AP. Chronic exposure of microglia to AP1.42 led to increased p38 M A P kinase expression and N F K B activation; in the presence of 4-AP, both factors were inhibited. Stimulation with Api.42 also led to the expression and production of pro-inflammatory mediators; 4-AP was effective in reducing the expression and production of these factors. Furthermore, 4-AP attenuated neurotoxicity induced by conditioned medium from AP1.42 stimulated microglia. In vivo, injection of Api_42 into rat hippocampus caused neuronal damage and increased microglial activation. Daily administration of 4-AP was found to suppress microglial activation and exhibited neuroprotection. These results suggest that 4- A P modulation of APi^-induced intracellular signaling pathways and functional responses in human microglia including microglial-mediated neurotoxicity serves as a potential therapeutic strategy in A D pathology. The chemokine C X C L 8 (IL-8) appears to potentiate AP1-42 responses in human microglia. RT-PCR and ELISA studies indicated that C X C L 8 potentiated APi^-induced expression and production of pro-inflammatory mediators; the expression of anti- inflammatory cytokines IL-10 and TGFPi remained unchanged from basal levels despite treatment with stimuli. Stimulation with C X C L 8 itself was effective in increasing microglial expression of pro-inflammatory mediators however, had no effect on protein levels of all these factors. C X C L 8 potentiation of APi-42-induced inflammatory mediators may have particular relevance to A D brain which exhibits elevated levels of the chemokine. T A B L E OF C O N T E N T S A B S T R A C T ii T A B L E OF CONTENTS iv. LIST OF FIGURES vii LIST OF TABLES ix ABBREVIATIONS x A C K N O W L E D G E M E N T S xiii Chapter 1: INTRODUCTION 1 1.1 A L Z H E I M E R ' S DISEASE: G E N E R A L 2 1.1.1 Early vs Late Onset A D 3 1.1.2 Processing of the Amyloid Precursor Protein 7 1.1.3 The Amyloid Peptide 11 •1.1.4 Cellular Dysfunction in A D 12 1.1.5 Amyloid Hypothesis and A D Pathology 14 1.1.5.1 Amyloid Hypothesis 15 1.1.5.2 Classification of Amyloid Plaques 16 1.2 ROLE OF I N F L A M M A T I O N IN A L Z H E I M E R ' S DISEASE 16 1.2.1 Inflammatory Response in Alzheimer's Disease '. 17 1.2.2 Inflammatory Polymorphisms Increase Risk of A D 19 1.2.3 Anti-Inflammatory Therapy as Treatment in A D 20 1.3 MICROGLIA A N D A L Z H E I M E R ' S DISEASE 21 1.3.1 Microglia 21 1.3.2 Role of Microglia in A D Pathogenesis 23 1.3.3 Ap as a Stimulus of Microglia 23 1.3.3.1 Receptors for Ap in Microglia 24 1.3.3.2 Stimulatory Effects of Ap on Microglia 25 1.3.4 Secretory Products of Microglia 27 1.3.4.1 Pro-Inflammatory Cytokines 27 1.3.4.2 Anti-Inflammatory Cytokines 30 1.3.4.3 Cyclooxygenase .....31 1.3.4.4 Chemokines 32 1.3.4.5 Cytotoxic Products 35 1.3.5 Signal Transduction Pathways in Microglia 40 1.3.6 Treatments for Alzheimer's Disease 41 1.3.7 Rationale for Proposed Research 43 1.4 R E S E A R C H HYPOTHESIS 49 1.5 S U M M A R Y OF R E S E A R C H OBJECTIVES 50 Chapter 2: M A T E R I A L S A N D METHODS 51 2.1 ISOLATION OF H U M A N MICROGLIA 51 2.2 ELECTROPHYSIOLOGICAL STUDIES OF H U M A N MICROGLIA A N D RECORDING SOLUTIONS 51 2.3 C A L C I U M SPECTROFLUOROMETRIC STUDIES 57 2.4 I M M U N O C Y T O C H E M I C A L STUDIES OF H U M A N MICROGLIA 58 2.4.1 Determination of p38 M A P Kinase and N F K B Immunoreactivity 58 2.4.2 Determination of COX-2 Immunoreactivity 60 2.5 REVERSE TRANSCRIPTASE P O L Y M E R A S E CHAIN REACTION OF H U M A N MICROGLIA 61 2.6 E N Z Y M E L I N K E D IMMUNOSORBENT A S S A Y 62 2.7 DETERMINATION OF FcyRII EXPRESSION IN H U M A N MICROGLIA 63 2.8 MICROGLIAL M O R P H O L O G Y A N D C E L L VIABILITY 64 2.9 DETERMINATION OF MICROGLIAL-MEDIATED NEUROTOXICITY 66 2.9.1 Preparation and Treatment of Human Fetal Microglia 66 2.9.2 Isolation of Primary Rat Hippocampal Neurons 68 2.9.3 Determination of Neuronal Damage from Microglial Conditioned Medium. 68 2.10 IN VIVO STUDIES OF Ap M 2 MEDIATED MICROGLIAL ACTIVATION A N D N E U R O N A L D A M A G E ; EFFECTS OF 4-AP ADMINISTRATION 69 2.10.1 Injection of APi_4 2 Into CA1 Region of Rat Hippocampus and Administration of 4-AP 69 2.10.2 Determination of Ap 1-42-Induced Neuronal Damage and Microglial Activation In Vivo 72 2.11 PEPTIDES A N D REAGENTS 73 2.11.1 Preparation of AP1-42 and Ap 42-i 73 2.11.2 Reagents. : 76 2.12 STATISTICAL METHODS 76 Chapter 3: EFFECTS OF A p M 2 O N M E M B R A N E POTENTIAL, M E M B R A N E CURRENTS and FcyRII EXPRESSION IN H U M A N MICROGLIA 77 3.1 R A T I O N A L E 77 3.2 RESULTS 80 3.2.1 Effect of AP1-42 on Membrane Current Expression; Voltage Clamp Studies. 80 3.2.1.1 The APM2-hiduced Outward Current is Attributed to K + 82 3.2.2 The Involvement of a G protein in the A P M 2 Induction of an outward K + current 87 3.2.2.1 Concentration-Dependent Inhibition of the GTPyS-Induced Outward K + Current by 4-AP and Several Other K + Channel Inhibitors 93 3.2.3 Molecular Biology Analysis of the Outward K +Current 95 3.2.4 Effect of A P M 2 on Membrane Potential of Human Microglia; Current Clamp Studies 98 3.2.5 Effect of Api-42 on FcyRII Receptor Expression 100 3.3 CONCLUSION 106 Chapter 4: MODULATION OF A p M 2 - I N D U C E D I N T R A C E L L U L A R SIGNALING A N D FUNCTIONAL RESPONSES OF MICROGLIA B Y 4-AMINOPYRIDINE: IN VITRO A N D INVIVO I l l 4.1 R A T I O N A L E I l l 4.2 RESULTS 113 4.2.1 Effect of Ap 1-42 on Intracellular Calcium of Human Microglia; Inhibition by 4-aminopyridine 113 4.2.1.1 Effects of A p M 2 on [Ca 2 +]i '. 113 4.2.1.2 Effects of 4-AP on Ap M 2 - induced C a 2 + influx pathway 119 4.2.2 Effects of AP1.42 on p38 M A P Kinase and NFtcB Activation in Human Microglia; Attenuation by 4-aminopyridine 122 4.2.3 Effects of Api.42 on Expression of Pro-Inflammatory Mediators; Attenuation by 4-aminopyridine 126 4.2.4 Effects of A0M2 on Production of Pro-Inflammatory Mediators; Attenuation by 4-aminopyridine 130 4.2.5 Effects of A P M 2 on microglial mediated primary hippocampal neuronal toxicity; inhibition by 4-aminiopyridine.... 135 4.2.6 In vivo effect of 4-aminopyridine on APi-42-induced neurotoxicity and microglial activation 138 4.2.6.1 4-AP reduces APi-42-induced neurotoxicity in vivo 138 4.2.6.2 4-AP reduces APi-42-induced microglial activation in vivo 141 4.3 CONCLUSION 143 Chapter 5: C X C L 8 (IL-8) POTENTIATION OF A p M 2 - I N D U C E D FUNCTIONAL RESPONSES OF H U M A N MICROGLIA 153 5.1 R A T I O N A L E 153 5.2 RESULTS 154 5.2.1 Effect of AP1.42, C X C L 8 (IL-8) Alone and Combined on Morphology of Human Microglia 154 5.2.2 Effects of C X C L 8 (IL-8) on APi_42-Induced Expression of Pro-inflammatory Mediators 156 5.2.3 Effects of C X C L 8 (IL-8) on APM2-Induced Production of Pro-inflammatory Mediators 160 5.3 CONCLUSION 166 Chapter 6: DISCUSSION OF THESIS R E S E A R C H A N D FUTURE DIRECTIONS 170 REFERENCES 174 L I S T O F F I G U R E S Figure 1-1. Processing of the Amyloid Precursor Protein Resulting in A(3 Formation 10 Figure 1-2. Simplified schematic illustrating the interaction between microglia and neurons of the CNS 39 Figure 1-3. Simplified schematic diagram illustrating the research presented in this thesis.. 48 Figure 2-1. Representative photomicrograph of microglia used in electrophysiological experiments 53 Figure 2-2. Intracellular application of GTPyS via the recording electrode 56 Figure 2-3. A representative photomicrograph of cultured human fetal microglia 65 Figure 2-4. Experimental procedure used to determine the effects of microglial conditioned medium on neuronal survival 67 Figure 2-5. The Dentate Gyrus of the Rat Hippocampus. 71 Figure 2-6. Representative photomicrographs of the AP1.42 preparation before and after heating 75 Figure 3-1. Typical current induced with acute application of AP1.42 to human microglia.... 81 Figure 3-2. Voltage-dependent activation of APi-42-induced outward current 83 Figure 3-3. Determination of the APi^-hiduced outward current's reversal potential via analysis of tail currents 85 Figure 3-4. The Ap^- induced outward K + current is inhibited by the non-selective K + channel inhibitor 4-AP 86 Figure 3-5. Typical profile of the intracellular GTPyS-induced outward current 88 Figure 3-6. The current induced with intracellular GTPyS and the current induced with extracellular A P i ^ 2 had similar thresholds for activation and reversal potentials 91 Figure 3-7. Effect of 4-AP (2 mM) on the intracellular GTPyS-induced outward K + current 92 Figure 3-8. Concentration-dependent inhibition of the intracellular GTPyS-induced outward K + current by 4-AP 94 Figure 3-9. Effects of AP1-42 on K v expression in human microglia 97 Figure 3-10. Effects of A p M 2 , A p 4 2 - i , High K + and PSS on V m of Human Microglia......... 99 Figure 3-11. Api^-induced expression of FcyRII : 101 Figure 3-12. A P M 2 induces an inward current in human microglia mediated by FcyRII activation 104 Figure 3-13. The APi-42-induced transient depolarization of human microglia is mediated by FcyRII activation 105 Figure 4-1. Acute application of APi_42 induces a slow, progressive increase in [Ca 2 +]i 114 Figure 4-2. In Ca2+-free solution, A p ^ 2 induces a small increase in [Ca2 +]j. 116 Figure 4-3. SKF96365, an inhibitor of SOC, had no effect on the increase in [Ca 2 +]i 118 Figure 4-4. The non-selective K + channel inhibitor, 4-AP, inhibits the APi-42-induced increase in [Ca 2 +]; 120 Figure 4-5. L a 3 + , an inhibitor of C a 2 + permeable channels, inhibits the Ap ^-induced increase in [Ca 2 +]i 121 Figure 4-6. Effects of 4-AP on APM2-induced p38 M A P kinase activation 123 Figure 4-7. Effects of 4-AP on Ap ^-induced N F K B activation 125 Figure 4-8. Effects of 4-AP on ApM2-induced expression of pro-inflammatory mediators 128 Figure 4-9. Effects of 4-AP on ApM 2-induced pro-inflammatory mediator production 131 Figure 4-10. Effects of 4-AP on APi^-induced COX-2 expressing microglia 134 Figure 4-11. Effects of microglial conditioned medium from microglia stimulated with A P i . 42, 4-AP each alone or in combination on neuronal survival 137 Figure 4-12. Effects of 4-AP on APM2-induced hippocampal neuron degeneration in vivo 140 Figure 4-13. In Vivo effects of 4-AP on Api-42-induced microglial activation 142 Figure 4-14. Summary of the effects of 4-AP on APi-42-induced intracellular signaling and functional responses in human microglia in this study 144 Figure 5-1. Effects of A P i ^ 2 , C X C L 8 (IL-8) each alone or in combination on morphology of human microglia 155 Figure 5-2. Effects of C X C L 8 (IL-8) on APi-42-induced pro inflammatory mediator and anti- inflammatory cytokine expression in human microglia 158 Figure 5-3. Effects of C X C L 8 (IL-8), A P M 2 each alone or in combination on pro- inflammatory mediator production 161 Figure 5-4. Effects of A P M 2 , C X C L 8 (IL-8) each alone or in combination on COX-2 expression in human microglia 165 L I S T O F T A B L E S Table 1-1. New Nomenclature for Chemokines (adapted from the IUIS/WHO Subcommittee on Chemokine Nomenclature, 2003) 33 Table 1-2. Summary of some secretory products of A(3 stimulated human microglia 38 Table 2-1. Primer Sequences for RT-PCR 62 Table 4-1. Fold increases in relative pro-inflammatory mediator mRNA induced by AP1-42, 4- AP, APi_42 + 4-AP, and AP42-1 compared to relative mRNA in control using semiquantitative RT-PCR 129 Table 4-2. Fold increases in pro-inflammatory mediator production induced by AP1.42, 4-AP, AP1-42 + 4-AP and AP42-1 compared to levels in control 132 Table 5-1. Fold increases in relative pro-inflammatory mediator mRNA induced by AP1-42, C X C L 8 (IL-8), Api-42 + C X C L 8 (IL-8) compared to relative mRNA in control 159 Table 5-2. Fold increases in pro-inflammatory mediator production induced by AP1.42, C X C L 8 (IL-8), Api_42 + C X C L 8 (IL-8) compared to levels in control 163 A B B R E V I A T I O N S A D Alzheimer's Disease AP amyloid beta AP1-40 40-residue C-terminal variant of amyloid beta A p M 2 42-residue C-terminal variant of amyloid beta A C T alpha-1 -antichymotrypsin A D A M a disintegrin and metalloproteinase AICD amyloid precursor protein intracellular domain A 2 M alpha-2-macroglobulin 4-AP 4-aminopyridine ApoE apolipoprotein E APP amyloid precursor protein ATP adenosine triphosphate B A C E P-site APP cleavage enzyme B B B blood brain barrier °C degrees Celsius C a 2 + calcium ion [Ca 2 +]i intracellular calcium concentration Caspases cysteine aspartyl proteases cDNA complimentary deoxyribonucleic acid CI" chloride ion CNS central nervous system C O X cyclooxygenase CR complement receptor CREB cAMP responsive element binding protein C-terminal carboxy-terminal CTF-a carboxy-terminal fragment alpha CTF-p carboxy-terminal fragment beta DAPI 4' -6' -diaminodino-2-phenylindole D M E M dulbecco's modified Eagle's medium D N A deoxyribonucleic acid ELISA enzyme linked immunosorbent assay EOFAD early onset familial Alzheimer's Disease ER endoplasmic reticulum E R K extracellular signal-regulated kinases Fc Fc receptor FPR formyl peptide receptor Fura-2/AM fura-2 acetoxymethylester G3PDH glyceraldehydes-3 -phosphate dehydrogenase GRO-a growth-related oncogene-alpha GTPyS guanosine 5'-0-(3-fhiotriphosphate) IFN-Y interferon gamma IL-lp interleukin-ip IL-4 interleukin-4 IL-6 interleukin-6 C X C L 8 (IL-8) interleukin-8 IL-10 interleukin-10 IL-13 interleukin-13 IFN-y interferon-gamma i.p. Intraperitoneal JNK c-jun N-terminal kinases K + potassium ion K v voltage-dependent potassium channel L a 3 + lanthanum ion L O A D late-onset Alzheimer's Disease LPS lipopolysaccharide LRP low density lipoprotein receptor related protein M A C membrane attack complex M A P K mitogen-activated protein kinase MCP-1 monocyte chemoattractant protein-1 M H C major histocompatibility complex M l P - l a macrophage inflammatory protein-1 alpha MIP-lp macrophage inflammatory protein-1 beta mRNA messenger ribonucleic acid mV millivolt N a 2 + sodium ion N A D P H nicotinamide adenine dinucleotide phosphate hydrogen N F K B nuclear factor kappa B N F T neurofibrillary tangle N M D A N-methyl-D-aspartate NO nitric oxide NPPB 5 -nitro-2-(3 - phenylpropylamino)-benzoate NSAID non-steroidal anti-inflammatory drug PBS phosphate buffered saline PCR polymerase chain reaction PFA paraformaldehyde P G E 2 prostaglandin E 2 P K C protein kinase C PPARy peroxisome proliferators activated receptor gamma PSEN1 presenilin-1 PSEN2 presenilin-2 PSS physiological salt solution R A G E receptor for advanced glycation end product RANTES regulated upon activation, normal T cells, expressed and secreted ROS reactive oxygen species RT-PCR reverse transcriptase polymerase chain reaction sAPPa secreted amyloid precursor protein alpha sAPPp secreted amyloid precursor protein beta S E M standard error of the mean s o c store-operated channel SOD superoxide dismutase SR scavenger receptor T E A tetraethylammonium TGFP transforming growth factor beta TNF-a tumor necrosis factor alpha V L D L - R very low density lipoprotein receptor V m membrane potential A C K N O W L E D G E M E N T S I would like to thank first and foremost Dr. J.G. McLarnon for giving me the opportunity to train in his laboratory and for his guidance, and supervision throughout the duration of my graduate work. I feel I have acquired a special set of skills from him that will be invaluable in my future endeavours. I am grateful to my committee members Dr. K . Dorovini-Zis, Dr. L. Kastrukoff and Dr. I. Laher for their assistance and invaluable insights into this work. I would like to acknowledge the following: Dr. S.U. K i m for providing human fetal microglia for my research, Hyun B . Choi and Jae K . Ryu for their enthusiasm, technical advice and suggestions, Dr. S. Jeong for his assistance with Fcyll receptor electrophysiology experiments, Dr. A . Klegeris for his assistance with Fcyll receptor experiments, Dr. D.G. Walker for his assistance in dissolving Ap peptide, Tariq Aziz and Ebrimma Gibbs for their assistance with the ELISA plate reader and to Dr. J. Church's Lab (Tony Kelly and Claire Sheldon) for providing primary neurons. I would also like to extend my gratitude to past students and.staff that have come through the lab: Dr. Hoon Bae, Vikram Goghari, Scott Hadland, Jeff Helm, Dr. Seok Ho Hong, Natinee Jarantonai, Andy Laycock, Prasongchai Sattayaprasert, Sandro Yong and Ladan Zand. A special thanks to the Faculty, Staff and students in the Department of Pharmacology and Therapeutics, U B C and to the Alzheimer Society of Canada for granting me a Doctoral Award (2001-2004). Most importantly, I would like to recognize my mother and father, my brother Dr. Luigi Franciosi, and my friends for their support and constant encouragement throughout my years as a graduate student. Xlll D E D I C A T I O N I would like to dedicate this thesis to my mother, father and brother Luig Chapter 1: INTRODUCTION Alzheimer's Disease (AD) is a progressive neurodegenerative disease marked by gradual cognitive decline and memory loss. Both the incidence and prevalence of A D increases sharply with age (Kawas et al., 2000; Jorm and Jolly, 1998). The German doctor Alois Alzheimer first reported the disease in 1907 after he examined a patient post-mortem who had died of an unknown mental illness. Alzheimer found unusual clumps of protein or plaques in the patient's brain at autopsy. The presence of plaques, which are composed of aggregated proteins called amyloid beta protein (Af3), and neurofibrillary tangles, which are intracellular aggregates of tau protein associated with neurofilaments, are the classical hallmarks of A D . In A D , amyloid deposition and neurofibrillary tangle formation are found predominantly in brain regions important for learning and memory processes. Most cases of A D are late-onset sporadic forms but 5-10% of cases are familial caused by a single mutation in genes located on chromosome 1, 14 and 21. The etiology of the late-onset sporadic form of A D is unknown but likely involves multiple factors. Among these are genetic polymorphisms (Roses, 1998), mutations of genes encoding the amyloid precursor protein (APP), presenilins and apolipoprotein E (APOE), physiological and environmental risk factors. The genetic defects all result in either an increase in total levels of Ap , or increase the more amyloidogenic form, APi_42- Overall, the neuropafhological changes and clinical symptoms of both familial and age-related forms of A D are similar. As noted above, A D is characterized by the presence of amyloid plaques and neurofibrillary tangles. Neuritic plaques in A D brain are also associated with gliosis and inflammation, which are characteristic pathological features of both familial and sporadic forms of A D . Reactive microglia and astrocytes are found in the vicinity of neuritic plaques with microglial activation preceding astrocyte activation (Frautschy et al., 1998). Microglia, the immune cells of the CNS, are activated by AP and it has been proposed that inefficient phagocytosis of the peptide by microglia could lead to hyperactivation of cells and release of inflammatory mediators and neurotoxic factors thereby contributing to neurodegenerative processes and subsequent cognitive impairment in A D (Akiyama et al.,2000). This introductory chapter provides an overview of A D , the inflammatory response in A D , the role microglia play in A D pathogenesis with particular emphasis on Ap as a stimulus of microglia, the secretory products of stimulated microglia and finally the hypothesis and research objectives of this thesis. 1.1 A L Z H E I M E R ' S D I S E A S E : G E N E R A L After the initial report in 1907 by Alois Alzheimer, intense research has focused on elucidating the risk factors, pathophysiologic abnormalities associated with A D and on mechanisms of impeding disease progression. A D is characterized by progressive memory loss, deterioration of cognitive function, progressive inability to carry out daily activities as well as psychiatric and behavioural disturbances which leads to death of the individual inflicted with this disease in a period of approximately ten years. A D accounts for 60% to 70% of cases of progressive cognitive impairment in elderly patients. The prevalence of A D doubles every 5 years after the age of 60, increasing from a prevalence of 1% among those 60- to 64-years-old to up to 40% of those aged 85 years and older (Von Strauss et al., 1999). Pathologically, A D selectively damages the frontal and temporal lobes, including the hippocampus—a structure involved in memory and visuospatial orientation (Arnold et al., 1991). In more advanced cases the pathology extends to other regions of cortex including the parietal and occipital lobes. This disease is distinguished from other dementias by the presence of amyloid plaques, neurofibrillary tangles and neuronal loss and is usually only diagnosed with certainty at autopsy. Amyloid plaques are extracellular deposits of insoluble, 8-10-nm amyloid fibrils that are polymers of Ap (Masters et al., 1985). Neurofibrillary tangles (NFT), contain paired helical filaments of abnormally phosphorylated tau protein that occupy the neuronal cell body and apical dendrites, in distal dendrites as neuropil threads, and in the abnormal neurites that are associated with some Ap plaques (neuritic plaques). The neuritic plaque consists of an extracellular core of amyloid fibrils intimately surrounded by dystrophic dendrites and axons, often containing paired helical filaments, as well as by activated microglia and reactive astrocytes (Cummings et al., 1998). Inflammation also plays a role in A D pathogenesis and seems to be facilitated by the presence of microglia, the immune cells of the CNS. 1.1.1 Early vs Late Onset AD A D is usually classified according to its age of onset. When the disease occurs before 65 years of age, it is classified as early-onset ("presenile") A D , while late-onset ("senile") A D (LOAD) occurs in subjects over 65 years of age. In addition to age, inheritance of predisposing risk factors appears to play a role in disease onset. Research suggests that early onset A D is familial (EOFAD), inherited as an autosomal dominant trait. EOF A D represent only about 5% of all A D cases. On the other hand, L O A D is more frequently sporadic, only a minority of these cases showing a clear family history and likely involves multiple factors most probably genetic susceptibility at multiple genes and interaction between these genes and/or environmental factors. Genetic Mutations Associated with Early Onset Familial AD Based on the observation that middle-aged patients with Down's syndrome (trisomy 21) commonly suffer from A D , researchers suspected that a gene on chromosome 21 could be involved in A D etiology. In 1987, linkage analysis in families with EOF A D identified a locus on chromosome 21 close to the APP gene (St. George- Hyslop et al., 1987). The APP protein is the precursor for Ap , which is the major component of amyloid plaques. Therefore, APP was considered an obvious candidate gene for A D . In 1991, missense mutations in the APP gene were identified (Chartier- Harlin et al., 1991; Goate et al., 1991) and the first gene involved in the etiology of A D was recognized. Approximately twenty mutations in the APP gene have been identified and all are clustered around the p- and y-secretase cleavage sites of the APP protein. These mutations result in an increase in the formation of AP40 and/or AP42, the more amyloidogenic and toxic species of Ap (Citron et al., 1992; Suzuki et al., 1994). The identification of APP as the first gene to be involved in A D led to the generation of transgenic animal models over-expressing normal or mutated human A D genes. Interestingly, most of the mouse models successfully mimic important features of the human disease, such as the presence of amyloid plaques, gliosis and neurodegeneration with age-related cognitive impairment, but neurofibrillary tangles are absent (Higgins and Jacobsen, 2003). Significant linkage of EOF A D to chromosome 14 was found and later identified as the presenilinl (PSEN1) gene as the responsible gene (Sherrington et al., 1995). Shortly after, evidence for a second gene from the presenilin family called presenilin 2 gene (PSEN2) was identified as being linked to EOF A D (Levy-Lahad et al., 1995). Presenilin activity is essential for the normal processing of APP (Citron et al., 1997). Transgenic mice over-expressing human wild-type or mutant PSEN1 have consistently shown elevated amounts of A P , especially Api^t2 (Citron et al., 1997) and double mutants (APP/PSEN1) produce more Ap than either transgene alone (Chapman et al., 2001). Moreover, the double transgenic mouse, PSEN2/APP, develops age-related cognitive decline associated with severe amyloidosis and inflammation in discrete brain regions (Richards et al., 2003). Mutations in the presenilins alter the y-secretase cleavage of APP resulting in over-production of the APi_42 peptides (Citron et al., 1997). Indeed, it has been suggested that the presenilinl protein could itself be a y-secretase (Wolfe et al., 1999). Overall, mutations in PS genes account for greater than 55% of all EOF A D mutations. Genetic Mutations Associated with Late Onset AD The apolipoprotein E (APOE) gene is recognized as a major risk factor for complex forms of A D , mainly in sporadic L O A D . By genetic linkage analysis using a collection of late-onset A D families, APOE was identified as a disease locus because of its localization in the peak linkage region on chromosome 19 (Pericak-Vance et al., 1991). However, only less than 50% of L O A D cases are carriers of the ApoE s4 allele, the genetic variant that predisposes to A D . In a study by Corder et al. (1993), a "dose- dependent" increase in the risk of A D from 20 to 90% and the mean age of onset decreased from 84 to 68 years was found with increasing number of s4 alleles. ApoE serves several functions involving the mobilization and redistribution of cholesterol during neuronal growth and after injury (Mahley, 1988), nerve regeneration, immunoregulation and activation of several lipolytic enzymes (Mahley and Rail, 2000; Vancea et al., 2000). The ApoE e4 allele increases amyloid deposition (Schmechel et al., 1993) however, the mechanism seems not to involve increased Ap production but rather stabilization and decreased clearance of fibrillar Ap deposits. Several other candidate genes have been linked to L O A D . On chromosome 12, genes encoding alpha-2macroglobulin (A2M), an acute phase protein, its receptor the low density lipoprotein receptor-related protein 1 (LRP1) and the transcription factor LBP- lc/CP2/LSF have been associated with L O A D . These genes play a role in AP metabolism (Narita et al., 1997; Pericak-Vance et al., 1997). Insulin degrading enzyme and urokinase plasminogen activator both located on chromosome 10 have also been associated with Ap degradation and clearance (Vekrellis et al., 2000; Ertekin-Taner, 2002). Increased risk of L O A D has also been associated with the very low density lipoprotein receptor (VLDL-R) gene, located on chromosome 9. V L D L - R expresses a cell-surface molecule specialized for the internalization of multiple ligands, including ApoE-containing lipoprotein particles, via clathrin-coated pits (Sakai et al., 1994). Polymorphisms for inflammatory genes including pro-inflammatory cytokines, A 2 M , and ai-antichymotrypsin (ACT) have also been associated with increased risk of developing A D (McGeer and McGeer, 2001). In the majority of L O A D cases, genetic factors act as predisposing agents; increasing the risk of disease above that of the general population. They probably interact with environmental factors or with other pathologic or physiologic conditions such as traumatic brain injury, cardiovascular abnormalities, oxidative damage and diet to exert their pathogenic effect. They may also interact between themselves to further enhance the probability of inducing the disease. 1.1.2 Processing of the Amyloid Precursor Protein Since changes in the generation or the degradation of Ap are, according to the amyloid hypothesis (discussed in Section 1.2.1.1), believed to trigger the molecular events in the pathogenic cascade of A D , research has been carried out to better understand the normal functions performed by APP and the physiological actions of Ap in order to more fully comprehend the disease process. The APP gene is localized to chromosome 21 and is expressed in many cell and tissue types including endothelial cells, glia and neurons (Schmechel et al., 1988). APP exists as three major protein isoforms APP770, APP751 and APP695, however, the APP695 is the predominant isoform in the brain. APP exists as a transmembrane protein with a large N-terminal ectodomain and a short intracellular C-terminus and can be localized to many membranous structures in the cell such as the endoplasmic reticulum and Golgi compartments, the cell membrane and further localized to postsynaptic densities, axons and dendrites (Schubert et al., 1991, Shigematsu et a l , 1992, Caporaso et al., 1994). The cloning and characterization of APP revealed that it possessed many features reminiscent of a membrane-anchored receptor molecule and subsequent work demonstrated that full-length APP functioned as a typical cell surface G-protein-coupled receptor (Okamoto et al., 1995) or as a secreted derivative that acts upon other cells. Neuronal APP colocalizes with Pi integrins at point contacts, suggesting a possible role in adhesion (Yamazaki et al., 1997). It has been demonstrated that APP binds directly to extracellular matrix molecules, particularly collagen type I (Beher et al., 1996). Furthermore, it has been reported that co-localization of APP with Piintegrin results in tyrosine kinase activation and subsequent productions of pro-inflammatory mediators in monocytes and microglia (Sondag and Combs, 2004). Other studies indicate roles of APP in cell motility (Sabo et al., 2001), synaptic transmission and plasticity (Perez et al., 1997; Dawson et al., 1999). The processing of APP involves three secretases (P, y, a) and two distinct intracellular metabolic pathways: the non-amyloidogenic and the amyloidogenic pathways (Figure 1-1). The a and p cleavages seem to be mutually exclusive events and each liberates a large extracellular domain of the protein, differing in size by only 17 amino acids at the carboxy terminus. In the non-amyloidogenic pathway, APP is cleaved, within the Ap sequence by a-secretase, a member of the A D A M family of metalloproteases. Cleavage of APP by a secretase generates extracellular soluble APP (sAPPa), a growth factor with neuroprotective and memory enhancing effects, as well as an intracellular C-terminal fragment (CTFa) corresponding to the 83 C-terminal amino acids of the protein (Oltersdorf et al., 1990). This a-secretase cleavage precludes the formation of Ap peptide. The activity of a-secretase is increased by activation of protein kinase C (PKC), perhaps via phosphorylation of sites on the intracellular carboxy- terminal tail of APP in turn making APP a more suitable substrate for a secretase. In the amyloidogenic pathway, Ap is generated by sequential cleavages of APP by P- and y-secretases. P-secretase, attributed to a single protein B A C E (P-site APP cleavage enzymes), is expressed in high amounts in neuronal tissue and is increased in A D (Vassar et al., 1999). Intracellularly, B A C E is localized primarily in the trans-Golgi network, endosomes, endoplasmic reticulum, and on the cell surface (Ling et al., 2003). The P- secretase cleavage generates sAPPp and CTFp corresponding to the 99 C-terminal amino acid of the protein. The CTFp is further cleaved by y-secretase, whose activity was shown to depend on the presence of a total of four components: presenilin, nicastrin, APH-1 and PEN-2 (Vassar et al., 1999; Edbauer et al., 2003). y-secretase activity generates the predominant Api_4o or AP1-42 fragments, as well as fragments spanning APi_39 and A P i ^ 3 , together with the remaining C-terminal tail (AICD). Similarly, after a cleavage, C83 can be processed by y-secretase to generate a shortened Ap-like fragment termed p3, plus the same C-terminal tail (AICD). In the absence of presenilin or nicastrin, the two C-teiminal fragments corresponding to C83 and C99 (CTF-a, P) accumulate and associate with the y-secretase complex at the plasma membrane where y- secretase is thought to be active. AICD is a very unstable peptide that upon ectopic expression in cultured cells may translocate to the nucleus and activate gene transduction (Gao and Pimplikar, 2001). Non-Amyloidogenic (No Plaque Formation) Amyloidogenic (Plaaue Formation) sAPPa neurotrophic neuroprotective Ap oligomer plaque formation AICD C83 C99 Figure 1-1. Processing of the Amyloid Precursor Protein Resulting in Ap Formation 1.1.3 The Amyloid Peptide Ap is a ~4 kDa protein. Amyloid fibrils are filamentous structures with a width of -10 nm and a length of 0.1-10 uM. Ap exists as monomers, dimers, and higher oligomers (3-6 units and up to 24 units), while further aggregation yields protofibrils and then fully-fledged fibrils that seem to compose the bulk of the amyloid plaques in A D brain (Walsh et al., 1999). In vitro experiments demonstrate that aggregation into fibrils is dependent on factors which include peptide concentration, acidic pH, environment, interaction with metals and various biomolecules and oxidative stress. The aggregation process results in the conversion of a Ap a helical filament or random coil into fibrils containing a p sheet conformation. Although A P i ^ o increases proportionately more in A D and may correlate better with synaptic change and cognitive deficits (Lue et al., 1999) , APi_42 aggregates more readily (Snyder et al., 1994) and is also more toxic (Pike etal., 1993). Although research has focused on the effects of Ap extracellularly, reports also indicate that A P can accumulate intracellularly particularly in neurons (Walsh et al., 2000) . It has been proposed that intracellular accumulation of Ap leads to neuronal lysis and dispersion of the cell contents into the surrounding parenchyma. According to this hypothesis, this extracellular Ap could then act as a locus for nucleation and aggregation of the soluble extracellular pool of Ap to form plaques (D'Andrea et al., 2001). Intracellular Ap has been found to co-localize with neurofibrillary tangles and is associated with signs of cytoskeletal degeneration. Furthermore, intraneuronal Ap accumulation has been associated with synaptic pathology and dysfunction (Takahashi et al., 2002; Oddo et al., 2003). 1.1.4 Cellular Dysfunction in AD Several mechanisms of A 0 mediated neurotoxicity have been postulated however the causal relationship and the sequence in which they occur is unknown; the mechanisms include inducing mitochondrial dysfunction, oxidative stress, disturbances in C a 2 + homeostasis, reactive oxygen species (ROS) generation, excitotoxicity, altered synaptic plasticity, ER stress and microglial activation (discussed in Section 1.3). Some of these mechanisms are described in further detail below. Mitochondrial abnormalities have been shown to be a very early pathological sign in A D (de la Monte et al., 2000). Several key mitochondrial enzymes including a- ketoglutarate dehyrogenase, pyruvate dehyrdogenase and cytochrome c oxidase are decreased in A D (Gibson et al., 1998; Kish et al., 1992; Mutisya et al., 1994). The APP molecule has been reported to interact with mitochondrial proteins resulting in mitochondrial dysfunction and inhibition of ATP synthesis (Anandatheerthavarada et al., 2003). Dysfunctional mitochondria are a source of reactive oxygen species (ROS) and inhibition of mitochondria has also been shown to increase the amyloidogenic processing of APP to Ap creating conditions for further cell damage (Gabuzda et al., 1994). Evidence for oxidative damage to proteins and D N A and increased levels of lipid peroxidation have all been described in postmortem tissue from patients with A D . Generation of ROS increases membrane lipid peroxidation resulting in impaired function of membrane ion-motive ATPases (Na + /K + - and Ca 2 +-ATPases), glucose and glutamate transporters leading to membrane depolarization and a decrease in cellular ATP levels (Mark et al., 1995; 1997). In addition to dysfunctional mitochondria, Ap may directly generate ROS or interact with other free radical generating systems such as the microglia and other cell types in the CNS via activation of N A D P H oxidase. The antioxidant enzyme superoxide dismutase (SOD) displays reduced activity in affected brain regions in A D (Zemlan et al., 1989). Ap alone directly interacts with metal ions such as copper (II) resulting in ROS generation. In addition, binding of Ap to copper (II), iron (III) and zinc (II) results in aggregation of the Ap peptide (Atwood et al., 1998) promoting plaque formation and toxicity. Increased levels of Ap have been shown to induce oxidative stress rendering neurons vulnerable to apoptosis and excitotoxicity through dysregulation of calcium homeostasis. The ability of antioxidants such as vitamin E to prevent impairment of the membrane transporters and to stabilize cellular calcium homeostasis supports a key role for membrane lipid peroxidation in disruption of calcium homeostasis by Ap (Goodman and Mattson, 1994). Studies of lymphocytes and lymphoblast cell lines from A D patients and age-matched normal control patients have documented alterations in cytokine and calcium signaling and increased levels of oxidative stress in immune cells from A D patients (Mattson, 2002). Furthermore, studies of the pathogenic actions of mutations in presenilins and APP that cause EOF A D have established central roles for perturbed cellular calcium homeostasis and oxidative stress in the neurodegenerative process. A direct effect of AP on intracellular calcium [Ca 2 T]; has been demonstrated. Whole cell patch clamp experiments of cultured neurons provided evidence that Ap can potentiate the activation of L-type voltage-gated calcium channels (Weiss et al., 1994; Ueda et al.,1997) rendering the neurons vulnerable to excitotoxicity (Mattson et al., 1992; Weiss et al., 1994). This toxicity was dependent on the Ap concentration used. As well, it has been reported that Ap can itself form calcium-conducting pores in cell membranes (Kawahara and Kuroda, 2000; Kagan et al., 2002). In addition, to its effects on plasma membrane calcium-regulating systems, Ap has been shown to have adverse effects on calcium regulation in the endoplasmic reticulum (ER) and mitochondria (Mattson and Chan, 2003). Moreover, it has been reported that increased amounts of [Ca 2 +]i induced by neuronal depolarization results in intraneuronal A P i ^ 2 production and subsequent neuronal death (Pierrot et al., 2004). Whether the mechanism of neuronal cell death in A D is necrotic or apoptotic remains unclear but could involve both processes. Apoptosis is believed to play a major role in A D pathology (Cotman and Anderson, 1995). Caspases (cysteine aspartyl proteases) initiate intracellular cascades leading to apoptotic outcomes which include proteolytic cleavage of cytoskeletal proteins and proteins of the nuclear scaffold. Apoptosis is initiated through intracellular mechanisms that often involve alterations in mitochondria or endoplasmic reticulum and by signaling through cell membrane death receptors. Some experimental studies suggest that Ap can activate caspases through binding of extracellular Ap to cell receptors (Dickson, 2004). Furthermore, accumulation of Ap in ER (endoplasmic reticulum) may activate apoptotic mechanisms through mitochondrial or ER stress (Lustbader et al., 2004). 1.1.5 Amyloid Hypothesis and AD Pathology A n ongoing debate in A D research relates to the etiology and connection between amyloid plaques and neurofibrillary tangles as well as their relative contribution to the disease. On the one hand, supporters of the "tau hypothesis" believe that the accumulation of hyperphosphorylated tau protein and resulting NFT formation leads to A D whereas supporters of the "amyloid hypothesis" believe that amyloid deposition is a central event and that amyloid plaques are the causative factor of A D . Although a number of studies have linked NFT numbers to synaptic loss and cognitive decline (Terry et al., 1991; McKee et al., 1991), Ap deposition and levels of Ap peptides are also correlated with cognitive decline (Naslund et al., 2000; Cummings et al., 1996). The amyloid hypothesis is more widely accepted since three mutations (APP on chromosome 21, PS1 on chromosome 14, and PS2 on chromosome 1), which underly EOF A D , lead to increased deposition of Ap (Scheuner et al., 1996). However, the amyloid hypothesis does not take into account reports which indicate that other factors such as oxidative damage, mitotic failure, neurofibrillary tangle formation and inflammation may occur upstream of amyloid formation and deposition. Improper metabolism of the amyloid precursor protein (as mentioned above in Section 1.1.2) with the resulting accumulation of the Ap fragment is viewed as the central event in A D pathogenesis (Selkoe, 2000). 1.1.5.1 Amyloid Hypothesis The hypothesis states that " A D is a pathological syndrome in which different gene defects can lead directly or indirectly to altered APP expression or proteolytic processing or to changes in Ap stability or aggregation. These result in a chronic imbalance between Ap production and clearance. Gradual accumulation of aggregated Ap initiates a complex, multistep cascade that includes gliosis, inflammatory changes, neuritic/synaptic change, tangles, transmitter loss and impaired cognitive function" (Selkoe, 2000). Key observations which support the amyloid hypothesis are summarized in Selkoe, 2000. 1.1.5.2 Classification of Amyloid Plaques Deposits of Ap that form in A D have been morphologically classified into four major types: diffuse plaques-most of the Ap peptide is not aggregated into a fibrillar structure, dystrophic neurites and paired helical filaments are infrequent or absent; primitive plaques- Ap is aggregated into amyloid and dystrophic neurites and paired helical filaments are clearly present; classic/neuritic plaques-Ap is highly aggregated to form a central core which is surrounded by a ring of dystrophic neuritis; compact plaques-consist of a solid core of amyloid but which lack a ring of dystrophic neuritis (Armstrong, 1998). Activated microglia are mainly associated with classic/neuritic plaques with few microglia associated with compact plaques. Two hypothesis exist for plaque formation: the life history hypothesis which states that plaque formation is thought to progress from diffuse through to compact and the "independent origin" hypothesis which states that plaques develop independently from each other. Irrespective of plaque progression, it has been reported that the relative frequency of these types of deposits changes during the progression of A D , with diffuse plaques being prevalent in the preclinical stages, and fibrillar plaques increasing in frequency as the disease progresses to clinical dementia (Dickson and Vickers, 2001; Thai et al., 2000). 1.2 ROLE OF INFLAMMATION IN ALZHEIMER'S DISEASE Whether inflammation is a cause and/or a consequence of A D pathology is still unknown. However, evidence continues to support the involvement of chronic inflammation in A D pathophysiology (Akiyama et al., 2000). Inflammatory processes in the CNS have the potential to be both beneficial and detrimental in A D pathogenesis (Wyss-Corray and Mucke, 2002). Interestingly, amyloid plaques with little or no inflammatory changes have been reported in the brains of non-demented individuals (Arriagada et al., 1992) which indicates that the plaques themselves are not responsible for clinical symptoms of A D but rather supports the involvement of inflammation in A D pathogenesis. Support for a role of inflammation in A D pathogenesis is also based on the association of inflammatory proteins, complement, potentially cytotoxic factors and microglia in the vicinity of neuritic plaques (discussed in Section 1.2.1). Also, polymorphisms of inflammatory factors associated with neuritic plaques are genetic risks factors for A D (discussed in Section 1.2.2). Moreover, support for a role of inflammation in A D is evident from epidemiological studies suggesting that anti-inflammatory drugs can prevent or slow the progression of A D (discussed in Section 1.2.3). 1.2.1 Inflammatory Response in Alzheimer's Disease Traditionally thought of as immunologically privileged, the CNS is known to have an endogenous immune system that is coordinated by immunocompetent cells such as the microglia. The inflammation associated with the CNS, neuroinfiammation, differs from that found in the periphery. The brain lacks pain fibers, making it difficult to recognize the occurrence of inflammation and the classic signs of inflammation such as rubor (redness), tumor (swelling), calor (heat), and dolor (pain) are typically not seen in the CNS. Also, the CNS differs from the periphery in that it is isolated from other organs by the blood-brain barrier (BBB). The B B B is formed by endothelial cells that are bound together by tight junctions surrounded by a thin basement membrane (i.e. basal lamina) which supports the ablumenal surface of the endothelium. Astrocytes are adjacent to the endothelial cell, with astrocytic end feet sharing the basal lamina preventing the entry of inflammatory cells, pathogens, and some macromolecules from blood into the brain. This barrier acts to protect neurons from the damages typically associated with inflammation. Contradictory results have been reported in both A D patients (Claudio, 1996; Caserta et al., 1998) as well as in animal models of A D (Podulso et al., 2001; Ujjie et al., 2003) as to whether the integrity of the B B B is disrupted or not. Regardless, neuritic plaques are the foci of local inflammatory responses, as evidenced by the presence of inflammatory mediators which include acute phase proteins (i.e. A C T , A 2 M , C reactive protein), cytokines, chemokines, cyclooxygenase, complement components and proteases (McGeer and McGeer, 1999; Eikelenboom et al., 1998; Akiyama et al., 2000). Ap peptide itself has been shown to induce a local inflammatory response since reports indicate that Ap binds to the complement factor CI and activates the classical pathway of the complement system (Rogers et al., 1992). The C3 and C5 fragments formed by complement activation, represent chemotactic and activation factors for microglia and astrocytes which have specific receptors for these fragments. The membrane attack complex composed of C5b, C6, C7, C8 and C9 can then affect surrounding cells cytotoxically or even cytolytically (McGeer and McGeer, 1999). Overactivation of the complement system is believed to play a role in A D pathogenesis since increases in complement protein (McGeer et al., 2000) and the presence of M A C (the membrane attack complex) on the surface of neurons have been detected in A D brain (Webster et al., 1997). Overactivation of complement could lead to amplification of an inflammatory response. The presence of pro-inflammatory cytokines including IL-1, IL-6, and TNF are of " particular interest since it has been shown in acute brain injury that these same cytokines may exacerbate lesion size and neuronal loss by their impact on already compromised neurons (Allan and Rothwell, 2001). These results would suggest that the presence of these cytokines could exacerbate the loss of neurons. Studies also indicate that some of these inflammatory proteins increase amyloid formation. The activated C l q complement component increases the aggregation of Ap and plays a role in Ap clearance by microglia (Webster et al., 1994; Rogers et al., 2002). Also, transgenic APP mice crossed with A C T transgenic mice had twice the amyloid load and plaque density compared with mice with the APP transgene alone (Nilson et al., 2001). Furthermore, it has been shown that IL-1 together with other cytokines can regulate APP synthesis and Ap production in vitro (Goldgaber et al., 1989; Blasko et al., 1999). 1.2.2 Inflammatory Polymorphisms Increase Risk of AD Polymorphisms for inflammatory genes including pro-inflammatory cytokines (IL-1, TNF-a, IL-6), A 2 M , and A C T have also been associated with increased risk of developing A D (McGeer and McGeer, 2001). The polymorphisms are in the noncoding regions of these genes (the promoter and untranslated regions). The polymorphisms are fairly common Ones in the general population so there is a strong likelihood that any given individual will inherit one or more of the high-risk alleles. Those alleles which favour increased expression of the inflammatory mediators are more frequent in A D patients than in controls. 1.2.3 Anti-Inflammatory Therapy as Treatment in AD Support for a role of inflammation in A D pathogenesis is also evident from epidemiological studies which indicate that non-steroidal anti-inflammatory drug (NSATD) use reduces the risk of developing A D , slows the progression and decreases the severity of dementia (McGeer et al., 1996; In 't Veld et al., 2001). It is generally accepted that NSAIDs exert their beneficial effects in A D via inhibition of C O X thereby inhibiting a wide range of inflammatory responses however, other mechanisms for NSAID action have been reported. These include activation of peroxisome proliferator- activated receptor y (PPARy), N F K B inhibition (Dodel et al., 1999), direct effects on amyloid processing (Weggen et al., 2003) and altering Ap aggregation (Thomas et al., 2001; Agdeppa et al., 2003). Differences exist in the efficacy of NSAIDs for use in treatment of A D and these differences may be due to the differing mechanisms of action of C O X inhibition (COX-2 vs COX-1/non-selective COX-1/2). Clinical results favouring the use of COX-1 inhibitors and/or mixed COX-1 /COX-2 inhibitors such as indomethacin and ibuprofen may be a result of COX-independent effects since these agents are capable of activating peroxisome proliferator-activated receptor gamma (PPAR-y), a nuclear receptor which has been shown to inhibit the expression of wide range of pro-inflammatory genes (Landreth and Heneka, 2001). The differing results of epidemiological studies versus recent clinical trials may also indicate that NSAIDs may not work in established A D , although for preclinical A D or mild cognitive impairment, a protective effect may be present (Aisen, 2002). Interestingly, NSAID treatment was associated with less in vivo microglial activation, as assessed by class II major histocompatibility complex (MHC) staining (Mackenzie and Munoz, 1998). NSAIDs suppressed Ap-stimulated proinflammatory and neurotoxic responses by microglia (Combs et al., 2000). Also, NSAIDs have been shown to reduce IFN-y/lipopolysaccharide induced microglial neurotoxicity (Klegeris et al., 1999). These studies collectively support the concept that inflammation mediated by microglia is an important component of A D pathophysiology, and strategies to control microglial activation could provide new therapeutic approaches for the treatment of A D . Studies using other anti-inflammatory agents such as low-dose prednisone for 1 year (Aisen et al., 2000) and hydroxychloroquine for 18 months (Van Gool et al., 2001) failed to demonstrate benefit in the treatment of A D . On the other hand, epidemiological studies have shown that estrogen replacement therapy is associated with improvement in cognitive performance, protection against cognitive decline and a decreased incidence of A D (Paganini-Hill, 1994; Costa et al., 1999). It has been reported that a possible mechanism of estrogen anti-inflammatory activity may be through inhibition of microglial activation (Bruce-Keller et al., 2000; Vegeto et al., 2001). 1.3 MICROGLIA AND ALZHEIMER'S DISEASE 1.3.1 Microglia Microglia, which are the immune cells of the central nervous system (CNS), were first identified and characterized by Pio del Rio-Hortega who published his findings in 1932 (del Rio-Hortega, 1932). Microglia represent approximately 10-20% of all glial cells in the CNS. Evidence from immunocytochemical studies using macrophage- specific markers have shown that microglia originate from monocytes entering the CNS at early stages of embryonic development (Perry et al., 1985; Ling and Wong, 1993). Other views include microglia as being derived from mesenchymal progenitor cells or that they are of neuroectodermal origin; the cells either originate from glioblasts or the germinal matrix (Kaur et al., 2001). Microglia are broadly classified based on two types of morphologies: ramified (resting) or ameboid (activated). In the resting state, microglia demonstrate a down-regulated process bearing phenotype however, are capable of becoming rapidly activated in response to pathological events. This would indicate that despite their apparent quiescence, microglia remain vigilant. Activated microglia demonstrate macrophage like properties: rounding of cells, lacking processes, upregulation of cell surface markers such as M H C Class II and complement receptors (indicative of their antigen presenting capacity) and often become reactive when carrying out phagocytosis (Kreutzberg, 1996; McGeer et al., 1988). In vitro, microglia possess an ameboid morphology immediately after isolation and then diversify into an inhomogenous population of both ameboid and ramified cells (Walker et al., 1995). Microglia respond quickly to a variety of signaling molecules and are typically the first cells to become activated at a site of injury (Gehrmann et al., 1995). Because of their responsiveness to an array of stimuli, microglia play a role in various immune functions in the brain which include host defence, neuroprotection, repair processes, phagocytosis, initiation and propogation of an inflammatory response (Banati and Graeber, 1994). Microglia express several cell surface receptors which couple stimuli to microglial functions including receptors for Fc and complement on their surface; t receptors implicated in microglial phagocytosis and cell lysis. A striking feature of microglial reactivity is the ability to synthesize and secrete a large number of substances, which, alone or in concert with factors derived from other brain or hematogenous cells, may have a crucial role in host defence or in the establishment or maintenance of brain damage. These secretory products include growth factors, cytokines, coagulation and complement factors, enzymes, reactive oxygen species and neurotoxins. Chronic activation of microglia leads to production of toxic substances which has been suggested as a factor contributing to neurodegenerative diseases (McGeer and McGeer, 1995). Moreover, 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 attraction of more microglia to the site of injury. 1.3.2 Role of Microglia in AD Pathogenesis Activated microglia cluster around amyloid plaques and microglial release products are thought to mediate a significant portion of neurotoxicity (Yates et al., 2000; Bamberger and Landreth, 2001; Benveniste et al., 2001; Combs et al., 2001; Meda et al., 2001). Factors released from dying neurons, such as ATP, could fuel cytokine release from microglia and aggravate consequences for neural cells. Thus, microglia play a significant role in the pathogenesis of A D ultimately leading to the cognitive decline which is associated with this disease. 1.3.3 Ap as a Stimulus of Microglia The presence of activated microglia in the vicinity of neuritic plaques may simply be a non-specific inflammatory response. However, this glial population appears to favor amyloid-containing plaques, indicating a specific interaction between Ap and microglia. Of note is the fact that activated microglia are associated with neuritic plaques containing dystrophic neurites and high levels of Ap (Rogers et al., 1988; Itagaki et al., 1989). In contrast, reactive microglia are not found within diffuse plaques, which are considered clinically benign Ap deposits. The clustering of microglia at neuritic plaques could be due to activation of microglia by chemotactic activation by Ap , activation by release products from damaged neurons or may be due to the presence of other pro-inflammatory mediators in the vicinity of plaques. 1.3.3.1 Receptors for A P in Microglia Several receptors in microglia have been proposed with varying abilities to bind fibrillar and nonfibrillar forms of Ap . They include the receptor of advanced glycation end products (RAGE) (Yan et al., 1996) and the scavenger receptor class A (SR-A) (El Khoury et al., 1996) and class B (SR-B) (Husemann et al., 2001). The R A G E receptor has been implicated in Ap induced secretion of pro-inflammatory mediators and chemotaxis (Lue et al., 2001b) and in the internalization of Ap by microglia (Yan et al., 1996). The SRs are thought to mediate adhesion and clearance of aggregated Ap , as well as the production of reactive oxygen species (Husemann et al., 2002). A cell surface receptor complex composed of ct6Pi integrin, CD47 and CD36 (an SR-B) also reportedly binds fibrillar Ap in microglia (Bamberger et al., 2003). The binding of Ap to this receptor complex results in the activation of tyrosine kinase-based signaling cascades and subsequent activation of microglia (McDonald et al., 1997; Combs et al., 1999). The formyl peptide receptor (FPR), which is coupled to a pertussis toxin-sensitive Gj-protein, calcium and phosphoinositide-specific phospholipase C, binds both soluble and fibrillar A p , mediates chemotactic effects of Ap as well as the production of pro-inflammatory mediators by microglia (Lorton et al., 2000). CD36, also involved in a receptor complex for Ap, mediates the binding of fibrillar Ap and activation of the downstream Src kinase family members, Lyn and Fyn, and the mitogen-activated protein kinase p44/42 (p44/42 M A P K ) resulting in reactive oxygen species and chemokine production (Moore et al., 2002). The serpin enzyme complex (Boland et al., 1996), heparan sulfate proteoglycans (Scharnagl et al., 1999), a 5Pi-integrin (Matter et al., 1998), LRP and insulin receptor have also been reported to bind Ap in microglia (Verdier et al., 2004). 1.3.3.2 Stimulatory Effects of Ap on Microglia There is extensive literature documenting the fact that Ap serves as a potent microglial activator; stimulation of microglia with Ap results in the expression (Lue et al., 2001a) and production of a repertoire of factors including proinflammatory cytokines such as interleukin (IL)-ip, tumor necrosis factor (TNF)-a, IL-6, the chemokines CCL3 (MIP la) , CCL2 (MCP-1) and C X C L 8 (IL-8) (Meda et al., 1996; 1999; Walker et al., 2001), the complement component C3 (Haga et a l , 1993), ROS (Meda et al., 1995) and glutamate (Klegeris et al., 1994; McDonald et al., 1997). Ap stimulation of microglia also leads to the production of excitotoxins, an unidentified neurotoxin and proteases (Giulian et al., 1995; 1997; Combs et al., 2001). It is believed that Ap alone is not toxic to neurons rather the microglia are necessary for neuronal killing in A D (Giulian et al., 1997). Furthermore, Ap stimulation of microglia enhances the expression of the co- stimulatory molecule CD40, a receptor which plays an important regulatory role in immune responses of microglia (Tan et al., 1999). Ap also acts as a chemotactic agent for microglia (Davis et al., 1992). Numerous researchers have described the ability of microglia to phagocytose and internally degrade Ap deposits (Frautschy et al., 1992; Shaffer et al., 1995), a process that may be important for plaque evolution. There is evidence that complement opsonizes Ap fibrils, facilitating their removal by microglial phagocytosis (Rogers et al., 1992). Surprisingly however, while microglia can clear even purified plaque material in vitro, plaque-removing activity is not obvious in human brain specimen. Microglia are believed to be overwhelmed and incapable of removing plaque material resulting in a continued inflammatory response, the emission of microglia-attracting and activating signals, and the release of potentially neurotoxic agents at high rates (Cotman et al., 1996). Ap can synergistically act with inflammatory mediators to enhance IL- ip , IL-6, TNF-a, CCL3 (MlP-la) , CCL4 (MIP-lp), and C X C L 8 (IL-8) production (Cooper et al., 2000; Yates et al., 2000). Ap-induced release of inflammatory mediators by microglia can then drive neurotoxic cascades that in turn recruit more microglia at the site of Ap plaques with subsequent amplification of inflammatory reactions. Interestingly, in cultures of mouse microglia and human monocytes, Ap did not alter the expression of the anti-inflammatory cytokine TGF-pi (Meda et al., 1999). These results would suggest that an imbalance between proinflammatory cytokine and possible inhibition could contribute to the pathogenesis of A D . The microglia themselves may be a source of Ap production in the A D brain. Although microglia have been shown to secrete Ap under the influence of Ap or pro- inflammatory stimuli (Bitting et al., 1996), microglia do not appear to express mRNA for APP in vivo (Scott et al., 1993). These results would indicate that microglia as a potential source of Ap is debatable and requires further research. The production of Ap by microglia would still be a small part of total Ap in the brain since the major source of AP production comes from neurons. Therefore, microglia cells are not necessary for the production of Ap plaques. However, evidence suggests that microglia assist in the conversion of non-fibrillar Ap to fibrillar Ap , and consequently, the development of neuritic plaques from diffuse plaques (MacKenzie et al., 1995; Sasaki et al., 1997). 1.3.4 Secretory Products of Microglia Microglial activation is accompanied by secretion of a plethora of substances (McGeer and McGeer, 1995). These include complement, acute phase proteins, cytokines, chemokines, prostaglandins, proteases, and reactive oxygen species. Since microglia are often the source and recipients of cytokine signals, autocrine loops may serve to maintain microglia in an activated state. Some of the secretory products of Ap stimulated microglia are discussed further below. 1.3.4.1 Pro-Inflammatory Cytokines Reactive glia produce a variety of molecules that trigger and contribute to chronic inflammation. Termed the "cytokine cycle", pro-inflammatory cytokines participate in a spectrum of cellular and molecular signaling that continuously feedback with potential neurodegenerative consequences (Griffin et al., 1998). IL-1B: In vitro, IL- ip has been shown to be produced by both rodent and human microglia and in response to Ap (Lue et al., 2001a; Meda et al., 1999). IL- ip release may in turn trigger production of other cytokines in an autocrine fashion (Benveniste et al., 1992). In particular, IL-1 has been reported to induce the expression and production of the chemokines M l P - l a and MEP-ip (McManus et al., 1998) and TNF-a in microglia (Chao et al., 1995). The activity of IL-1 is blocked by a naturally occurring receptor antagonist (IL-lra) that binds to the Type I IL-1 receptor, but does not initiate signal transduction. In turn, secretion of IL-1 by microglia can have profound effects on astrocytes since in addition to the induction of a variety of inflammatory and cytotoxic mediators, IL-1 acts as a mitogen for rodent astrocytes (Giulian et al., 1994) and induces a stress response in human astrocytes (Lee et al., 1995). IL- ip has also been directly implicated in neuronal degeneration (Griffin and Mrak, 2002). In A D , overexpression of IL- ip is a consistent feature found within cortical regions from post-mortem brains. Double-labeling immunohistochemical studies have localized IL- ip to plaque-associated microglia (Griffin et al., 1989; Griffin et al., 1995). IL- ip overexpression appears to occur early in plaque evolution evident in diffuse, non- neuritic AP deposits (Griffin et al., 1995). IL- ip in particular may be involved in the growth of dystrophic neurites around plaques (Sheng et al., 1996), excessive production and processing of amyloid precursor protein as well as the synthesis of most of the known plaque associated proteins (Akiyama et al., 2000). Furthermore, IL-1 p has been linked to increased acetylcholinesterase resulting in decreased acetylcholine (Li et al., 2000) thereby contributing to cognitive decline. TNF-a: Microglia are a prominent source of TNF-a in the brain. Depending on the stimulus, even a short exposure can be very effective and result in a strong and lasting release response (Hanisch, 2002). Thus, in CNS insults, rapid induction of microglial TNF-a production could critically influence subsequent events. Microglia can also release soluble TNF-R (sTNF-R), an antagonist of circulating TNF-a, and hence affect the available TNF-a pool and probably regulate potential TNF-a consequences. While TNF-a seems to play a central role in glial functions (Benveniste, 1992), its effect on neuronal cell viability is controversial both in vitro (Cheng et al., 1994; Chao and Hu, 1994; Giulian et al., 1993) and in vivo (Gary et al., 1998; Barone et al., 1997). However, reports indicate that TNF-a is essential for Ap-induced neurotoxicity (Viel et al., 2001). TNF-a has autocrine actions in microglia since in addition to acting as a stimulus of microglia, it also induces proliferation of microglia (Dopp et al., 1997; Thery and Mallat, 1993) and enhances IFN-y-induced nitric oxide production (Merrill et al., 1993). Although, TNF-a is regarded as a pro-inflammatory cytokine, some down-regulating effects on microglia have been reported such as inducing the expression and release of the anti-inflammatory cytokine IL-10 (Sheng et al., 1995). Immunohistochemical studies show an increase in microglial TNF-a localized to senile plaques, suggesting its participation in Ap-induced inflammation (Dickson et al., 1993). As well, TNF-a is elevated in the serum, CSF and cerebral cortex of A D patients (Fillit et al., 1991; Tarkowski et al., 1999). TNF-a reportedly induces neuronal production of AP1.42 (Blasko et al., 2001) which would then lead to increased amyloid plaque formation. IL-6: Microglia release IL-6 in early phases of CNS insults (Raivich et al., 1999). Subsequently, IL-6 may act on astrocytes to involve these cells in the orchestration of attempts for tissue repair (Raivich et al., 1999). Interestingly, IL-6 can have both pro- and anti-inflammatory outcomes (Raivich et al., 1999). On the one hand, like IL-ip and TNF-a, IL-6 is considered a proinflammatory cytokine with actions which include initiating and coordinating inflammatory responses, inducing acute phase proteins, increasing vascular permeability, lymphocyte activation and antibody synthesis (Akiyama et al., 2000). As well, IL-6 is autocrine in microglia since it is a major mediator of microglial activation (Raivich et al., 1996; Streit et al., 2000). On the other hand, there is also evidence that IL-6 may also have anti-inflammatory actions; for example, IL-6 can inhibit glial production of TNF-a induced by IFN-y, IL- lp and the bacterial endotoxin lipopolysaccharide (LPS) (Benveniste, 1992). IL-6, as well as IL-1 P, have been suggested to modulate APP synthesis (Vandenabeele and Fiers, 1991). Plasma IL-6 concentrations in A D subjects have been found to be significantly elevated compared to control cases (Shibata et al., 2002). Histologic studies have demonstrated IL-6 immunoreactivity co-localized with diffuse plaques lacking neuritic pathology (Hull et al., 1996) and a strong immunohistochemical staining for IL-6 within senile plaques of A D patients (Strauss et al., 1992). Increased levels of IL-6 mRNA are expressed in glial cells surrounding amyloid plaques in A D transgenic mice (Mehlhorn et al., 2000; Tehranian et al., 2001). IL-1 and IL-6 could promote the synthesis and processing of APP, thus inducing further Ap production and deposition (Buxbaum et al., 1992; Mrak and Griffin, 2000). 1.3.4.2 Anti-Inflammatory Cytokines Interleukin-4 (IL-4), -10, -13, and transforming growth factor-P (TGF-p) share features of anti-inflammatory, immunosuppressive and neuroprotective actions. Much of these outcomes can be attributed to a downregulation of microglial production of cytokines, e.g., IL- lp and TNF-a, or the attenuation of their secondary release effects. For example, IL-10 has been reported to inhibit IL-6 production in microglia (Heyen et al., 2000). IL-4 and IL-13 also interfere with IL-1 bioactivity by enhancing IL-IRa synthesis (Dinarello, 1997a, 1997b). In addition, these cytokines can alter microglial cell surface marker expression (Chao et al., 1993; Suzumura et al., 1994; Raivich et al., 1999; Sawadaetal., 1999). TGF-P, a factor with multiple biological activities in many cells and playing roles in various tissue developments and immune responses, reduces proinflammatory cytokine and chemokine production. TGF-p is also a potent chemoattractant for microglia (Yao et al., 1990). Most notably, TGF-p has been reported to reduce A D plaque load in an animal model of A D through a phagocytotic mechanism mediated by microglia (Wyss- Coray et al., 2001). Moreover, TGF-P i has been shown to be protective against neuronal cell damage (Flanders et al., 1998). 1.3.4.3 Cyclooxygenase Cyclooxygenase (COX) catalyses the formation of prostanoids comprised of prostaglandins, prostacyclin, and thromboxanes from arachidonic acid and is a major target of non-steroidal anti-inflammatory drugs (NSAIDs). C O X exists in two isoforms: constitutive (COX-1) and inducible (COX-2). COX-2 is rapidly expressed in several cell types in response to growth factors, cytokines, and pro-inflammatory molecules and is considered a major player in inflammatory reactions and instrumental in neurodegenerative processes of several acute and chronic diseases such as A D . The concept of a pathogenic role of C O X in A D is derived from epidemiological studies reporting an association between long-term NSAID use and reduced risk of A D (discussed in Section 1.2.3), although not every investigation has proved the same protective effect (Aisen, 2002). Histological analyses of C O X levels in A D brains have produced conflicting results. Several studies reported increased neuronal COX-2 immunoreactivity compared to control brain tissues (Yasojima et al., 1999) whereas other studies report a decrease in the number of COX-2-positive neurons with increasing severity of dementia (Yermakova et al., 2001; Hoozemans et al., 2002a). However, elevated levels of prostaglandin E2 (PGE2), a product of C O X activity, have been reported in CSF of A D patients relative to control (Ho et al., 2000; Montine et al., 1999). Interestingly, PGE2 downregulates microglial activation, self-limiting the inflammatory process (Minghetti and Levi, 1998). 1.3.4.4 Chemokines Chemokines belong to a superfamily of small (8-14 kDa) secreted proteins that were initially identified as regulators of leukocyte trafficking during inflammatory responses. A table summarizing the new nomenclature of chemokines is presented in Table 1-1. The new chemokine nomenclature uses CC, C X C , X C or CX3C, indicating the class to which the chemokine belongs, followed by the letter " L " (for ligand) and then a number which corresponds to that already in use to designate the genes encoding each chemokine. Table 1-1. New Nomenclature for Chemokines (adapted from the IUIS/WHO Subcommittee on Chemokine Nomenclature, 2003) Systematic Name CXC Chemokines CXCL1 CXCL2 CXCL3 CXCL4 CXCL5 CXCL6 CXCL7 CXCL8 CXCL9 CXCL10 CXCL11 CXCL12 CXCL13 CXCL14 CXCL15 CXCL16 C Chemokines XCL1 XCL2 CX 3C Chemokines CX3CL1 Original Ligand Name (Human) GROa GROP GROy PF4 ENA78 GCP-2 NAP-2 IL-8 Mig JJMO I-Tac SDF-la/p BCA-1 BRAK/bolekine Unknown Lymphotactin/ SCM-la/ATAC SCM-lp Fractalkine CC Chemokines CCL1 1-309 CCL2 MCP-1/MCAF/TDCF CCL3 MTP-la/LD78a CCL3L1 LD78p CCL4 MJP-lp CCL5 RANTES CCL6 unknown CCL7 MCP-3 CCL8 MCP-2 CCL9/10 Unknown CCL11 eotaxin CCL12 Unknown CCL13 MCP-4 CCL14 HCC-1 CCL15 HCC-2/Lkn-l/MIP-18 CCL16 HCC-4/LEC/LCC-1 CCL17 TARC CCL18 DC-CK1/P ARC/AMAC-1 CCL19 MIP-3 p/ELC/exodus-3 CCL20 MIP-3a/LARC/exodus-l CCL21 6Ckine/SLC/exodus-2 CCL22 MDC/STCP-1 CCL23 MPIF-l/CKp8/CKp8-l CCL24 Eotaxin-2/MPIF-2 CCL25 TECK CCL26 Eotaxin-3 CCL27 CTACK/ILC CCL28 MEC Microglia can produce several chemokines including CXCL1 (growth-related oncogene-alpha: GRO-cc), CCL3/4 (macrophage inflammatory protein-1 alpha/beta: MIP- loc/(3), CCL2 (monocyte chemoattractant protein-1; MCP-1), CCL5 (regulated upon activation, normal T cells, expressed and secreted: RANTES) and C X C L 8 (interleukin-8: IL-8) in response to experimental stimulation by bacterial agents, Ap peptides, as well as cytokines, such as TNF-a and IL-1 (Ehrlich et al., 1998; Peterson et al., 1997; Janabi et al., 1998). This would suggest that activated microglia could serve in further microglial recruitment. Via chemokines, microglia can also affect neurons and astrocytes and orchestrate the migration of leukocytes. Stimulation of microglia with chemokines results largely in the recruitment and migration of microglia to sites of injury. More recently, the functional repertoire of chemokines has expanded to include fundamental roles in other cellular processes such as survival and proliferation of neurons and glia (Ambrosini and Aloisi, 2004). The chemokine CX3CL1 (fractalkine), occurring in soluble and membrane-bound forms, is unique in that it is predominantly expressed on neurons, whereas the CX3CL1 receptor CX3CRI mainly associates with microglia (Harrison et al., 1998; Nishiyori et al., 1998). Therefore, disturbances in CX3CL1 levels may be sufficient to trigger or enhance microglial activation. Neuronal CX3CL1 may not only support microglial survival (Boehme et al., 2000), it also decreases microglial activation (Zujovic et al., 2000). CX3CL1 can induce microglial migration (Maciejewski-Lenoir et al., 1999). Moreover, microglia are also a source of CX3CL1 (Zujovic et al., 2000), allowing for autocrine signaling in these cells. Under normal CNS conditions, low levels of chemokines are detected. However, under pathological conditions such as in A D , levels of chemokines are elevated (Xia et al., 1999). Elevated levels of C X C L 8 (IL-8) (Galimberti et a l , 2003) and C X C L 8 (IL-8) receptors (Xia et al., 1997) have been detected in A D brain. Also, increased CCL2 (MCP-1; Ishizuka et al., 1997) and CCL4 (MIP-ip; X ia et al., 1998) immunoreactivity is evident in A D and localized to plaque associated microglia. Interestingly, the expression of the chemokine receptors CCR3 and CCR5 was also increased in plaque associated microglia which could explain how microglia are strongly recruited to sites of plaque pathology. 1.3.4.5 Cytotoxic Products In addition to secretion of an unidentified neurotoxin in response to Ap (Giulian et al., 1995) as well as pro-inflammatory cytokines (TNF-a, IL- ip ; Giulian et al., 1993; Griffin and Mrak, 2002), activated microglia are capable of releasing several potentially cytotoxic factors in vitro including reactive oxygen intermediates, NO, proteases, arachidonic acid derivates, excitatory amino acids and quinolinic acid. In either human or rat brain, activated microglia are the most abundant source of oxygen free radicals. Large amounts of superoxide anions (O2") are generated on the microglial external membrane and subsequently released into the surroundings (Klegeris and McGeer, 1994; Colton and Gilbert, 1987). Activated microglia are also capable of producing hydrogen peroxide (H2O2) and reactive nitrogen intermediates (Hensley et al., 1998). H2O2 released by activated glia, though itself fairly innocuous, can be altered by peroxidases to form hypochlorous acid (HOC1), which is highly toxic to cells. Peroxynitrite (ONOO) formed through the interaction of NO with O2" can result in both nitration of tyrosines and nitrosylation of cysteines within both enzymes and structural proteins. Activation of microglial and ensuing ONOO" production has been linked to AP toxicity in cultured neurons (Xie et al., 2002). Whereas physiological levels of N O may influence synaptic efficacy by regulating neurotransmitter release (Brenman and Bredt, 1997; Garthwaite, 1991), excess N O is toxic to neurons. However, the cytotoxic properties of microglia are subject to considerable species variation. For example, NO production is established for rat, but not in human microglia (Colton et al., 2000; Lee et al., 1993). In addition, iNOS was not found in stimulated human microglia (Zhao et al., 1998; Walker etal., 1995). Proteases contribute in various functions of microglia. For example, cathepsin E and cathepsin S, endosomal/lysosomal proteases, have been shown to play important roles in the major histocompatibility complex (MHC) class Il-mediated antigen presentation of microglia by processing of exogenous antigens and degradation of the invariant chain associated with M H C class II molecules, respectively (Nakanishi, 2003). Some members of cathepsins are also involved in neuronal death after being secreted from microglia and clearance of phagocytosed Ap peptides. Tissue-type plasminogen activator, a serine protease, secreted from microglia participates in neuronal death, enhancement of N M D A (N-methyl-D-aspartate) receptor-mediated neuronal responses, and activation of microglia to contribute to further neurodegeneration (Tsirka, 2002; Nakanishi, 2003). Derivatives of APP, including Ap, can stimulate glutamate release from microglia (Barger and Basile, 2001; Klegeris and McGeer, 1997). Glutamate is an excitatory amino acid. Glutamate neurotoxicity mediated by activation of N M D A receptors and subsequent Ca influx has been suggested to be involved in neurodegeneration in A D (Koh et al., 1990; Mattson et al., 1992). N M D A receptor antagonists are efficacious neuroprotectants (Piani et al., 1992). Similarly, quinolinic acid, also a N M D A agonist, is an endogenous neurotoxic metabolite of the tryptophan-kyneurine pathway and is produced predominantly by microglia in response to stimuli including Ap and IFN-y (Guilleman et al., 2003; 2005). Binding of quinolinic acid to N M D A receptors leads to neurodegeneration through both excitotoxic (Stone, 1993) and oxidative (Santamaria et al., 2001) mechanisms. A table summarizing the secretory products of human microglia stimulated with Ap is presented in Table 1-2. A schematic diagram illustrating the interactions between microglial secretory products and surrounding neurons is shown in Figure 1-2. Table 1-2. Summary of some secretory products of Ap stimulated human microglia. Cytokines IL-1 a (Veerhuis et al., 2003) IL- lp (Lue et al., 2001a; Nagai et al., 2001) IL-8 (Lue et a l , 2001a; Nagai et al., 2001) IL-6 (Lue et a l , 2001a) TNF-a (Lue et al., 2001a; Nagai et al., 2001) Chemokines M l P - l a (Lue et al., 2001a; Nagai et a l , 2001) MCP-1 (Lueetal., 2001a) Complement C3 (Veerhuis et al., 1999) Cytotoxic Products Quinolinic Acid (Guilleman et al., 2003) Superoxide Anion (Lue and Walker, 2002) Growth Factors macrophage colony stimulating factor (M-CSF) (Lue et al., 2001a) nerve growth factor (NGF) (Heese et al., 1998) Figure 1-2. Simplified schematic illustrating the interaction between microglia and neurons of the CNS Healthy neurons may inform microglia about their normal activity by releasing factors such as the chemokine fractalkine. Resting microglia could support neuronal function and survival by production of neurotrophic factors. Upon transformation of microglia from resting to activated in response to surrounding stimuli such as Ap, microglia may also produce cytokines and other factors (reactive oxygen intermediates) that are potentially toxic to neurons as well as induce further recruitment of microglia (adapted from Hanisch, 2002). 1.3.5 Signal Transduction Pathways in Microglia In vitro studies have shown that exposure of microglia to A(3 results in activation of a host of intracellular signaling pathways linked to effector functions of microglia such as secretion of inflammatory and neurotoxic substances. The tyrosine kinases Lyn, Syk, and F A K as well as p38 and E R K members of M A P K were first shown to be activated on exposure of microglia to A P resulting in the generation of superoxide radicals (McDonald et al., 1997; 1998). In particular, Ap stimulated the rapid, transient activation of extracellular signal-regulated kinase 1 (ERK1) and ERK2, p38 M A P K and downstream M A P K effectors RSK1 and RSK2 and phosphorylation of the transcription factor c A M P response element-binding (CREB) protein, providing a mechanism for Ap-induced changes in gene expression. Also, it was reported that Ap activation of Lyn and Syk initiated a signaling cascade resulting in a transient release of intracellular calcium, activation of P K C and the calcium-sensitive tyrosine kinase P Y K 2 resulting in the production of neurotoxic substances, cytokines and reactive oxygen species (Combs et al., 1999). In addition to Lyn and Syk, activation of the transcription factor N F K B was required for T N F - a production by microglia (Combs et al., 2001). Other signaling pathways such as the CD36-dependent signaling cascade involves, Lyn and Fyn (also a tyrosine kinase), and the M A P K p44/42 resulting in reactive oxygen and chemokine production and microglial migration (Moore et al., 2002). Moreover, the Ap-induced phosphorylation and translocation of M A R C K S (Myristoylated alanine-rich C kinase substrate) and MARCKS-related protein (MRP), proteins implicated in membrane-cytoskeletal alterations underlying microglial adhesion, migration, secretion, and phagocytosis, were reportedly dependent on a tyrosine kinase- PKC-delta signaling pathway in microglia. (Murphy et al., 2003; Nakai et al., 2001). The expression and secretion of IL-1 P by Ap stimulated microglia involves rapid activation of three different M A P K s (p38, ERK1/2, and INK) and N F K B activation (Kim et al., 2004; Kang et al., 2001). Furthermore, IL- lp production and chemotaxis of microglia has been linked to activation of a G protein coupled receptor and calcium' increase (Lorton et al., 1997; Tiffany et al., 2001). Moreover, induction of an outward K + current in rat microglia attributed to activation of voltage-dependent Kv l .3 and Kv l .5 channels (Chung et al., 2001) and increases in [Ca2 +]j have been reported in response to Ap stimulation (Silei et al., 2000; Korotzer et al., 1995). 1.3.6 Treatments for Alzheimer's Disease The most widely used therapy for A D is the acetylcholinesterase inhibitors which lead to increased acetylcholine and subsequent reduction in cognitive impairment associated with this disease. Although acetylcholinesterase inhibitors are helpful in treating the symptoms of the disease, they have no effect to prevent or delay disease progression. Other current therapeutic approaches include antioxidants, NSAIDs to reduce inflammation (discussed in Section 1.2.3), N M D A receptor antagonists, methods of inhibiting amyloid generation and aggregation as well as increasing amyloid removal. The secretases that produce Ap are considered potential therapeutic targets for the treatment of A D . Treatment of mice with a y-secretase inhibitor resulted in reduced A p levels in the brain and attenuated Ap deposition (Dovey et al., 2000). Compounds with effects to block y-secretase without blocking other y-secretase targets, which could be lethal, have been developed (Petit et al., 2001) and are currently being tested in clinical trials. Epidemiological data indicate that cholesterol may play a role in A D pathogenesis. Retrospective studies of individuals taking H M G - C o A (P-hydroxy- P- methylglutaryl-coenzyme A) reductase inhibitors or statins show a large reduction in the risk for developing A D (Wolozin et al., 2000). In vitro and in vivo studies indicate that statins and other cholesterol-lowering agents decrease Ap levels and Ap deposition (Refolo et al., 2001; Simons et al., 1998), whereas high-cholesterol diets in APP transgenic mice increase Ap deposition (Refolo et al., 2000). In vitro, statins seem to promote a favourable shift toward the non-amyloidogenic pathway of APP processing leading to increased secretion of the neurotrophic sAPPa peptide and decrease in Ap production (Buxbaum et al., 2001; Puglielli et al., 2001). These results would indicate that cholesterol lowering drugs may be of therapeutic benefit in the prevention and treatment of A D . Active immunization with synthetic Ap^2 has been shown to be effective in transgenic models of A D to significantly reduce Ap plaques (Schenk et al., 1999; Bard et al., 2000). Using the approach of APi_42 immunization, specific Ap^2 antibodies are elicited and these antibodies move across the B B B resulting in the removal of amyloid plaques and improved cognitive performance (Kotilinek et al., 2002). Although the mechanism through which antibodies to Ap decrease plaque burden is not clear it is believed to involve the disruption of Ap fibrils, prevent Ap fibril formation, block the toxic effects of Ap aggregates and enhance clearance of plaques by microglia (Golde, 2003). In a recent clinical trial, administration of the vaccine in patients with A D resulted in discontinuation of the trial after signs of meningoencephalitis developed in about 6% of the treated patients. The follow-up in patients receiving the vaccine showed that 60% produced antibodies against amyloid-containing plaques and patients with higher antibody titer had a slowing in cognitive loss (Hock et al., 2002; 2003). Due to the side effect observed in A D patients receiving the vaccine, Ap vaccines with differing doses of Ap peptide/anti-Ap peptide adjuvants are being explored. As well, alternative Ap vaccination methods such as the Ap gene vaccination method have been proposed (Qu et al., 2004). In my study, I have used the approach of blocking inflammation by inhibiting microglial mediated inflammatory responses using a modulator of Ap-induced intracellular signaling pathways in these cells. 1.3.7 Rationale for Proposed Research A chronic inflammatory reaction exists in A D brain (Akiyama et al., 2000). Activated microglial cells are localized to neuritic plaques and are a significant source of inflammatory mediators (McGeer and McGeer, 1995). Thus, selective modulation of microglial signaling pathways could be an effective strategy to lessen inflammation in A D . The activation process induced by Ap in microglia has been associated with specific cell surface receptors (discussed in Section 1.3.3.1) and intracellular signaling pathways (discussed in Section 1.3.5) which include membrane K7 current expression, altered intracellular calcium, activation of tyrosine kinases, p38 M A P kinase and activation of transcription factors (NFKB, C R E B ) . These intracellular signaling pathways are intimately linked to particular cellular functions of microglia (discussed in Section 1.3.3.2) which include proliferation, phagocytic activity and motility. In terms of actions on bystander cells such as neurons a critical cellular role of microglia is the production of neurotoxins (discussed in Section 1.3.4.5). Therefore, it is likely that microglial inflammatory responses in A D are dependent on a complex pattern of altered membrane currents, intracellular calcium and changes in other intracellular signal transduction pathways. Therapeutic approaches focused on modulation of stimulus-induced signaling and microglial-mediated inflammatory responses could serve as a therapeutic strategy in neurodegenerative diseases. As noted above, changes in membrane current patterns are an important signaling pathway in microglia. Based on these results, pharmacological modulation of activated channels could be a novel and effective maneuver to inhibit microglial mediated inflammatory responses. My preliminary data indicated that acute application of AP1.42 to human microglia induced a novel outward current consistent with activation of a K7 channel. As well, a novel finding in this work was that intracellular application of the non-hydrolyzable analogue of GTP, GTPyS, induced an outward K + current similar in properties to the outward K + current induced with acute A0M2- Based on these results, I used the non-selective K + channel inhibitor, 4-aminopyridine (4-AP), to investigate properties of this current. Preliminary experiments showed that 4-AP acted to inhibit the outward current induced by acute APi_42- This finding led to a detailed investigation of 4- AP as a pharmacological modulator of AP-stimulated microglia. Interestingly, 4- aminopyridine has been previously used as a therapy in treatment of A D patients (Wesseling et al., 1984; Davidson et al., 1988). This compound passes through the B B B whereas other non-selective K + channel blockers such as T E A do not (Soni and Kam, 1982). Since 4-AP blocked the channel upregulated by acute Api-42,1 wanted to carry out further experiments as to whether 4-AP would be an effective modulator of other signaling pathways and functional responses of human microglia. As pointed out above, 2*1" • Ca along with other second messengers are important signaling factors in microglia. Work done by the Landreth laboratory (Section 1.3.5) indicated that an increase in intracellular Ca was involved in response to Ap in rodent microglia in addition to the activation of several other intracellular signaling factors such as tyrosine kinases, members of M A P K and N F K B (McDonald et al., 1997; 1998; Combs et al., 2001). Many of these second messengers are linked to cytotoxic secretory products of microglia. I wanted to investigate the effects of Ap on Ca and other intracellular signal transduction factors such as p38 M A P K and N F K B in human microglia and whether 4-AP modulated these second messengers. Some studies were also undertaken to investigate i f the Fc receptor might be involved in response to the Ap peptide. Ap stimulated microglia produce a host of factors implicated in inflammation and neurodegeneration as stated above (Griffin et al., 1998). The pro-inflammatory cytokines IL- ip , IL-6 and TNF-a and the inducible enzyme COX-2 are important modulators of inflammatory processes (Benveniste, 1992; O'Neill and Ford-Hutchinson, 1993) and increased levels of these factors have been observed in A D brain (Griffin et al., 1998; Pasinetti and Aisen, 1998; Ho et al., 1999). It was therefore of interest to determine in this research program whether 4-AP had effects to modulate the expression and production of these pro-inflammatory mediators. I then carried out a series of studies to examine 4-AP actions in vivo. Since in vitro results suggested that 4-AP is a potential modulator of A P i ^-induced intracellular signaling pathways and functional responses in human microglia, 4-AP was tested for potential anti-inflammatory and neuroprotective effects in vivo. The effects of 4-AP administration on microglial activation and neuronal toxicity induced by a local injection of APi_42 into the rat hippocampus was investigated. In a separate set of studies, I also examined i f the pro-inflammatory effects of Ap could be potentiated by another stimulus of microglia, the chemokine C X C L 8 (IL-8). The rationale for this study was that in a thorough gene expression study, C X C L 8 (IL-8) was reported as the most prominent factor expressed by Ap stimulated human microglia (Walker et al., 2001). Moreover, increased levels of C X C L 8 (IL-8) have been observed in brains of A D patients (Galimberti et al., 2003). Since C X C L 8 (IL-8) is secreted (Ehrlich et al., 1998) by stimuli such as Ap (Nagai et al., 2001) and in turn C X C L 8 (TL-8) stimulates chemotaxis of microglia (Cross and Woodroofe, 1999), C X C L 8 (IL-8) is autocrine in these cells. Potentiation of Ap induced responses by stimuli such as C X C L 8 (IL-8) would be relevant to inflammatory actions of microglia in A D brain. In summary, my research program will determine the effects of APi_42 on the intracellular signaling pathways (ion channel expression, Ca , p38 M A P K , N F K B ) and functional responses induced by Ap (expression and production of pro-inflammatory factors, neurotoxicity), both in the presence and absence of the modulator 4-AP. As well, the in vivo effects of 4-AP will be detenriined on APM2-induced microglial activation and neurotoxicity. Furthermore, the potentiating effects of the chemokine IL-8 on A P M 2 - induced functional responses in human microglia will be investigated. A model of the research presented in this thesis is shown in Figure 1-3. STIMULUS A p M 2 + C X C L 8 / Ion Channel microglia 4-AP modulation 4 FUNCTIONS Release Agents: ( I L - i p I IL-6 N C X C L 8 I TNF-a ^ Neurotoxins, others In Vivo Figure 1-3. Simplified schematic diagram illustrating the research presented in this thesis. Overall, some intracellular signaling pathways and functional responses shown in the diagram were examined in \ \ \ \ A 2 stimulated human microglia. Some evidence suggests that Fc receptors may be involved in Ap actions. 1.4 RESEARCH HYPOTHESIS Microglia are involved in inflammatory responses that mediate neuronal degeneration associated with Alzheimer's Disease (AD). Signaling pathways in microglia regulate cellular functional responses both under normal and pathological conditions. The hypothesis of the proposed research is that changes in membrane currents, [Ca 2 +]i, intracellular signal transduction pathways are involved in the signaling pathways of A p M 2 and are coupled to microglial functional responses including secretion of substances that would potentiate an inflammatory response as well as lead to neurotoxicity both in vitro and in vivo. Additionally, modulation of these signaling pathways could serve a site for potential therapeutic intervention in decreasing microglial-induced neuronal toxicity and subsequent neuronal loss associated with A D . One such agent, the non-selective K + channel blocker 4-AP, has been studied in detail as a modulator of Ap-stimulated human microglia. 1.5 S U M M A R Y O F 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 pharmacological modulation by 4-AP of A P i -42-induced intracellular signaling pathways and functional responses including peptide-induced microglial mediated neurotoxicity. The specific objectives are listed below: 1. to identify APi .42-induced Ca signaling pathways, second messenger activation and alterations in membrane potential and currents in human microglia. 2. to investigate 4-AP as a modulator of Api-42-induced membrane currents, intracellular calcium [Ca 2 +]i levels, second messengers (p38 M A P K , N F K B ) and for effects on functional responses of human microglia such as expression and production of inflammatory mediators ( T N F - a , IL- ip , IL-6, C X C L 8 (IL-8) and COX-2) and potential neurotoxicity. 3. To determine the effects of 4-AP in vivo on Ap ^-induced microglial activation and neurotoxicity. 4. To determine the potentiating effects of C X C L 8 (IL-8) on A p i .42-induced expression and production of inflammatory mediators (TNF-a, IL- ip , IL-6, C X C L 8 (IL-8) and COX-2). The specific objectives 1 to 4 are presented in thesis chapters 3 to 5. Chapter 2: MATERIALS AND METHODS 2.1 ISOLATION OF HUMAN MICROGLIA Human microglia were prepared according to procedures reported previously (Satoh et al., 1995). Briefly, embryonic brain tissues 12-18 weeks gestation were incubated in phosphate-buffered saline (PBS) containing 0.25% trypsin and DNase (40 ug/mL) for 30 min at 37°C. Enzyme treated tissues were dissociated into single cells by gentle pipetting. Dissociated cells were then cultured into Dulbecco's Modified Eagle's Medium (DMEM) containing 5% horse serum, 5 mg/mL glucose, 20 ng/mL gentamicin and 2.5 ug/mL amphotericin B. After 7-10 days of growth in culture flasks, freely floating microglia were collected from a medium of mixed cell cultures and plated on glass coverslips. The purity of the microglial cultures was in excess of 98% as determined by immunostaining with the cell specific markers C D l l b or ricinus communis agglutinin-1. Use of embryonic human tissues was approved by the Clinical Screening Committee for Human Subjects of the University of British Columbia. 2.2 ELECTROPHYSIOLOGICAL STUDIES OF HUMAN MICROGLIA AND RECORDING SOLUTIONS Procedures used in whole-cell patch clamp studies have been described previously (McLarnon et al., 1997). Briefly, one-day plated coverslips were placed on the stage of an inverted microscope (Nikon TMS). I used one-day plated coverslips since microglia plated for more than five days express an inactivating outward K7 current in response to a depolarizing step not present in cells one to two days post-plating (McLarnon et al., 1997). Coverslips were plated at low density since only one cell/coverslip was used and only large ameboid cells were chosen since they were easier to patch. Typical density of coverslips and ameboid morphology of microglia used in whole cell patch clamp experiments is shown in Figure 2-1. A n amplifier (Axopatch 200B, Axon Instruments, Foster City, CA) was used to record macroscopic currents as well as membrane potential (V m ) . Patch pipettes were fabricated using Corning glass no. 7052 with resistances in the range of 2-4 M Q . Capacitance and series resistance were compensated manually on the amplifier. The whole-cell configuration was used and data sampled at 5 kHz with the low-pass filter set at 1 or 2 kHz. Figure 2-1. Representative photomicrograph of microglia used in electrophysiological experiments. Microglia cells one-day post-plating in normal bath solution displayed both ramified (elongated, spindle-shaped) and ameboid (rounded) morphologies. Coverslips were plated at low density and only large ameboid cells (indicated with arrows) were used in whole-cell patch clamp experiments. (xlOO magnification; scale bar = 20 jim). Protocols used in voltage clamp experiments were generated by computer and consisted of applying a depolarizing step from a holding potential (VH) of -60 mV in order to study outward K + currents. A l l voltage clamp experiments in Chapter 3 were carried out with 500 u M 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) in bath solution in order to remove any chloride contribution to the overall whole cell current. Some current clamp experiments were also carried out to determine the effects of A(3i^2 on V m . Both resting membrane potential recordings and stimulus-induced changes in V m were recorded in these experiments. Data were recorded on disk and analyzed off-line using pClamp 6.0 software. A l l experiments were performed at room temperature (20- 22°C). The normal bath solution contained (in mM): NaCl (140), KC1 (5), CaCl 2 (1), MgCb (1), glucose (10), HEPES (10); pH adjusted to 7.3. The pipette solution contained (in mM): KC1 (140), NaCl (10), M g C l 2 (1), E G T A (0.5), ATP (1), HEPES (10); pH adjusted to 7.3. In experiments where elevated extracellular K + was applied, 40 m M KC1 was used to replace an equivalent amount of NaCl. Stimuli were applied in the bath solution and perfused onto the cell using a gravity fed system with the exception of GTPyS which was applied intracellularly through the recording electrode (discussed below; Chapter 3). In order to determine whether a G protein was involved in inducing an outward K + current in microglia as has been previously shown in macrophages (McKinney and Gallin, 1992), the non-hydrolyzable analogue of GTP, GTPyS (10 u,M), was applied intracellularly through the recording electrode (Figure 2-2). Upon rupture of the cell membrane in the whole-cell patch clamp mode, GTPyS diffuses from the electrode into the cell where it irreversibly activates G protein coupled receptors to induce second messenger signaling in microglia. Patch pipette- GTPyS Whole-cell configuration Cell- Figure 2-2. Intracellular application of GTPyS via the recording electrode. Upon rupture of the cell membrane in the whole-cell patch clamp mode, GTPyS diffuses into the cell where it exerts its intracellular effects. 2.3 CALCIUM SPECTROFLUOROMETRIC STUDIES Levels of intracellular calcium [Ca 2 +]i in human microglia were monitored using calcium spectrofluorometry. Cells plated on glass coverslips were loaded with 1 u M fura-2/AM (Molecular Probes, Eugene, OR) with the solubilizing agent 0.02% pluronic acid in standard physiological solution (PSS). The PSS solution contained (in mM): NaCl (126), KC1 (5), M g C l 2 (1.2), CaCl 2 (1), D-glucose (10) and HEPES (10) at pH 7.4. 9+ 9+ In experiments performed with no Ca in the extracellular solution, a Ca -free PSS was used where the CaCl 2 was replaced with 1 m M EGTA. Following the wash period in dye-free solution, the coverslips were mounted on the stage of a Zeiss Axiovert inverted microscope containing a x 40 quartz objective lens. The cells were then exposed to alternating wavelengths of 340 and 380 nm U V light at intervals of 6 seconds and emission light was passed through a 510 nm filter (bandwidth of 20 nm). The signals were acquired from a digital camera (DVC-1300 Camera, Photometries) and recorded using an imaging system (Empix, Mississauga, ON) as fluorescence ratios of 340/380 9+ • every 6 seconds. Increases in [Ca ]j were expressed as 340/380 fluorescence ratios since unherent uncertainties exist in conversion of ratios into [Ca ]i using an in vitro calibration (Takahashi et al., 1999). Stimuli were applied acutely using a micropipette and solutions exchanged using a vacuum suctioning system. A l l experiments were carried out at room temperature (20-22°C). 2.4 IMMUNOCYTOCHEMICAL STUDIES OF HUMAN MICROGLIA 2.4.1 Determination of p38 MAP Kinase and N F K B Immunoreactivity The effects of stimuli on phosphorylated p38 M A P kinase (phospho-p38) in human microglia were determined using immunocytochemistry according to a similar procedure described previously in microglia (Tikka and Koistinaho, 2001). Briefly, following pre-incubation in serum-free medium for 48 hrs, cells were treated for 30 min with AP1-42 (5 uM), 2 m M 4-AP alone, or AP1.42 in combination with 4-AP following a 30 min preincubation with 4-AP, and AP42-1. After treatment, cells were fixed with 4% paraformaldehyde (PFA) in 0.1 M PBS, washed in PBS and permeabilized in 0.2% Triton X-100 containing 5% NGS in 0.1 M PBS/0.5% B S A (BPBS) solution for 25 min. The cells were then incubated in rabbit anti-human phospho-p38 (1:250 dilution, Cell Signaling) containing 5% NGS in BPBS at 4°C for 48 hrs. As a negative control, the phospho-p38 primary antibody was omitted. Following wash in PBS, cells were incubated with Alexa Fluor 488 anti-rabbit IgG secondary antibody (1:100, Molecular Probes, Eugene, OR, USA) containing 5% NGS in BPBS at room temperature for 2 hr. Following a wash in PBS, cells were incubated in 4'-6'-diaminodino-2-phenylindole (DAPI, Molecular Probes) at 1 ug/mL in PBS to visualize nuclei and determine cell numbers in the field of view. Cells were then washed in water and mounted onto glass slides using gelvatol, examined under a Zeiss light microscope and photographed using a cooled C C D camera. Cells positively stained with phospo-p38 M A P kinase were determined from four representative fields in four independent experiments (approximately 600 cells/treatment group) and the ratio of phosho-p38 positive cells to total number per field was determined (at x200 magnification). As a positive control, a similar procedure was carried out with cells stimulatd with LPS for 30 min in three independent experiments. Values are expressed as means ± S E M and statistical significance was determined using one-way A N O V A and Newman-Keuls multiple comparison post-test (p < 0.05). In resting cells, the transcription factor N F K B is retained in the cytoplasm in an inactive form by binding to a family of inhibitory molecules: IKBCC or IKBP (Christman et al., 2000). Activation of N F K B results in rapid phosphorylation and degradation of IKBS allowing the active complex p65/p50 of N F K B to be released, translocate to the nucleus and transactivate target genes. To investigate the effect of 4-AP on APi_42-induced N F K B activation, a similar immunocytochemical procedure as described for the determination of p38 M A P K activation was used to determine the effects of 8 hr stimulation with ApM2 (5 u,M), 4-AP alone, A P M 2 in combination with 2 m M 4-AP following a 30 min preincubation with 4-AP, 4-AP alone and AP42-1 on the nuclear translocation of p65 as has been described previously in microglia (Nakajima et al., 1998). The primary antibody used for this set of experiments was rabbit anti-human p65 (1:250 dilution, Santa Cruz, CA) , in order to target the active p65 N F K B subunit and cells with intense staining localized to the nucleus were considered positively stained cells. Results were obtained from four representative fields in five independent experiments (approximately 600 cells/treatment group) and summarized as mean ± S E M . Positive control experiments consisted of stimulating microglia with LPS for 12 hrs and determining the number of positive p65 cells as described above in six independent experiments. As a negative control, experiments were carried out with p65 primary antibody omitted. 2.4.2 Determination of COX-2 Immuhoreactivity COX-2 production was determined in microglia using immunocytochemistry. Following 48 hr pre-incubation in serum-free medium, cells were stimulated for 24 hrs with stimuli. In experiments where 4-AP was used as a modulator, cells were pre- incubated with 4-AP for 30 min prior to addition of stimuli and 4-AP was maintained in solution subsequent to addition of stimuli. After treatment, cells were fixed with 4% PFA in 0.1 M PBS, washed in PBS and permeabilized in 0.2% Triton X-100 containing 10% goat serum in 0.1 M PBS/0.5% B S A (BPBS) solution for 20 min. The cells were then incubated in rabbit anti-human COX-2 (1:200 dilution, Cayman Chemical, Ann Arbor, MI) containing 10% goat serum in BPBS at 4°C for 72 hrs. As a negative control, the COX-2 primary antibody was omitted. Following wash in PBS, cells were incubated with Alexa Fluor 488 anti-rabbit IgG secondary antibody (1:100, Molecular Probes, Eugene, OR, USA) containing 5% goat serum in BPBS at room temperature for 1 hr. Following a wash in PBS, cells were incubated in DAPI (Molecular Probes) at 1 ug/mL in PBS to visualize nuclei and determine cell numbers in the field of view. Cells were then washed in water and mounted onto glass slides using gelvatol, examined under a Zeiss light microscope and photographed using a cooled C C D camera. The number of cells positively stained with COX-2 was determined (at x200 magnification) and expressed as a ratio of COX-2 positive cells to total number from four representative fields (approximately 150 cells/field). As a positive control, cells were treated with LPS for 12 hrs and the number of COX-2 positive cells determined as described above in three independent experiments. 2.5 REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION OF HUMAN MICROGLIA Human fetal microglia were seeded into poly-L-lysine coated 12-well plates at a density of approximately 5 x 104 cells/well. Following pre-incubation in serum-free conditions for 48 hrs in order to promote a resting state, human microglia were then treated for 8 hours with stimuli. In experiments where 4-AP was used, cells were pre- incubated with 4-AP for 30 minutes prior to addition of stimuli and subsequently maintained in solution following addition of stimuli. Total R N A was isolated using TRIzol (GIBCO-BRL, Gathersburg, MD), subjected to DNase treatment and then processed for the first strand complimentary D N A (cDNA) synthesis using Moloney murine leukemia virus (M-MLV) reverse transcriptase (GIBCO-BRL). cDNA products were then amplified by PCR using a GeneAmp thermal cycler (Applied Biosystems, Foster City, Ca). The PCR reaction buffer included cDNA, sense and antisense primers and Taq polymerase. Specific sense and antisense primers with the expected product size are listed in Table 2.1. PCR consisted of an initial denaturation step of 95°C for 6 minutes followed by a 30-40 cycle amplification program consisting of denaturation at 95°C for 35 s, annealing at 59°C for 1 min and elongation at 72°C for 1 min. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used as a reaction standard. The amplified PCR products were identified by electrophoresis using 1.5% agarose gels containing ethidium bromide and then visualized under U V light. The intensities of each band were measured by densitometry using the NIH Image J 1.24 software (National Institutes of Health, Bethesda, Maryland, USA). The band intensities of PCR products in control and with stimuli were measured and expressed as relative mRNA levels (mRNA values normalized to G3PDH). Table 2-1. Primer Sequences for RT-PCR Product Sequence Size (b.p.) COX-2 sense 5' -TTC 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 ' 305 COX-2 antisense 5' - A G A T C ATCTCTGCCTGAGTATCTT-3 ' IL- ip sense 5 ' -AAAAGCTTGGTGATGTCTGG-3 ' 179 IL- ip antisense 5' -TTTC A A C A C G C A G G A C A G G - 3 ' IL-6 sense 5' - G T G T G A A A G C A G C A A A G A G G C - 3 ' 159 IL-6 antisense 5' - C T G G A G G T A C T C T A G G T A T A C - 3 ' C X C L 8 sense 5' - A T G A C T T C C A A G C T G G C C G T G - 3 ' 301 C X C L 8 antisense 5 ' - 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 - 3 ' TNF-a sense 5' -C A A A G T A G A C C T G C C C A G A C - 3 ' 490 TNF-a antisense 5 ' - G A C C T C T C T C T A A T C A G C C C - 3 ' IL-10 sense 5' - A G A T C T C C G A G A T G C C T T C A G C A G A - 3 ' 194 IL-10 antisense 5' -CCTTGATGTCTGGGTCTTGGTTCTC-3 ' TGFpi sense 5' -TTGC AGTGTGTTATCCGTGCTGTC-3 ' 185 TGFPi antisense 5 ' -C A G A A A T A C A G C A A C A A T T C C T G G - 3 ' K v l . l sense 5' -GTTAGGGG A A C T G A C G T G G A - 3 ' 482 K v l . l antisense 5' -CTGAGC A G G A G A G G A A A C C A G - 3 ' K v l . 2 sense 5' - G G G A C A G A G T T G G C T G A G A A - 3 ' 513 K v l . 2 antisense 5' - G G A G G A T G G G A T C T T T G G A C - 3 ' Kv l . 3 sense 5 ' - G C G A C G A G A A G G A C T A C C C - 3 ' 513 . Kv l .3 antisense 5 ' - T G C T G C T G A A A C C T G A A G T G - 3 ' K v l . 5 sense 5' -G A G G A C G A G G A G G A A G A A G G - 3 ' 528 Kv l .5 antisense 5' -C A A G C A G A A G G T G A T G A T G G - 3 ' K v l . 6 sense 5' -GGGAGTC A G G A G G A A G A G G A - 3 ' 569 K v l . 6 antisense 5' - A T G C T G G G A A A A A G C G A A T - 3 ' Kv2.1 sense 5' - A C A G A G C A A A C C A A A G G A A G A A C - 3 ' 385 Kv2.1 antisense 5' -C A C C C T C C A T G A A G T T G A C T T T A - 3 ' Kv3.1 sense 5 ' - T C C T G A A C T A C T A C C G C A C G - 3 ' 620 Kv3.1 antisense 5' -GAACTCTACCTTGTTGGGGC-3 ' G3PDH sense 5' -CC ATGTTCGTC A T G G G T G T G A A C C A-3 ' 251 G3PDH antisense 5' -GCC A G T A G A G G C A G G G A T G A T G T T C - 3 ' 2.6 ENZYME LINKED IMMUNOSORBENT ASSAY ELISA kits ( R & D Systems, Minneapolis, M N , USA) were used to determine the production of TNF-a, IL-6, IL- ip and C X C L 8 (IL-8) in culture supernatants from human fetal microglia stimulated for either 24 hrs (in the case of C X C L 8 (EL-8) potentiation of A P M 2 effects; Chapter 5) or 48 hrs (in the case of 4-AP modulation of AP1-42 effects; Chapter 4) after pre-incubation in serum-free medium for 48 hrs. In experiments where 4-AP was used as a modulator of A p ^ 2 effects, cells were incubated with 4-AP for 30 min prior to addition of stimuli and maintained in solution following addition of stimuli. The kits were able to detect low levels of the cytokines (as low as 4.4 pg/mL of TNF-a, 1 pg/mL IL- lp , 0.7 pg/mL IL-6 and 10 pg/mL C X C L 8 (IL-8)). After incubation of microglia with stimuli, cell culture supernatants were collected and stored at -70°C. 2.7 DETERMINATION OF FcyRII EXPRESSION IN HUMAN MICROGLIA Microglia were plated into 96-well culture plates (1 x 105 cells/well) in 200 jol of D M E M containing 5% horse serum. Api^2 was applied in duplicate wells. Cells were treated and incubated in 5% C 0 2 , 95% air at 37°C with A p i ^ 2 for 30 min and 48 hr following pre-incubation in serum-free medium for 48 hrs. Expression of Fey receptor type II (FcyRII) was measured following procedures described previously (Klegeris et al., 2000). In brief, cells were fixed by air-drying overnight and then blocked with 3% B S A in PBS at room temperature for 2 hr. Subsequently, the plates were incubated with monoclonal FcyRII antibody (1:500 dilution in blocking solution) for 1 hr. In negative control experiments, the FcyRII primary antibody was omitted. Following incubation with FcyRII antibody, cells were briefly washed four times in PBS and then incubated with goat anti-mouse IgG alkaline phosphatase conjugate diluted 1:4000 in blocking solution for 1 hr. After incubating the cells with substrate buffer containing 1 mg/ml of Sigma 104 phosphate substrate in 0.1 M diethanolamine buffer, pH 9.8, optical densities (OD) were measured every 10 min for 1 hr using a 405-nm filter. The OD of the negative control well was then subtracted from the OD of untreated and treated wells at the corresponding time point. The change in OD over 60 min in treated samples was normalized to that of untreated. Values were obtained from n= 4 independent experiments for 30 min treatment and n=6 independent experiments for 48 hr treatment; each experiment was repeated in duplicate. 2.8 MICROGLIAL MORPHOLOGY AND C E L L VIABILITY Human microglia were pre-incubated in serum-free medium for 48 hrs prior to addition of stimuli in.these studies (immunocytochemistry, RT-PCR, ELISA, FcR, neurotoxicity) in order to promote a resting state. This procedure yields a homogenous population of cells with a ramified, process-bearing morphology. Following pre- incubation in serum-free medium, microglia were exposed to inflammatory agents for periods of up to 48 hrs which often yielded a rounding of cells lacking processes indicative of an ameboid morphology. Our laboratory routinely uses DAPI staining to examine the viability of microglia with different treatments (Choi et al., 2003) and I found no evidence of nuclear condensation or fragmentation under any of the experimental conditions indicating no loss of cell viability in the present experiments. Figure 2-3. A representative photomicrograph of cultured human fetal microglia. Both ramified and ameboid microglia were present one-day post-plating (at x 100 magnification; scale bar = 50 urn). 2.9 DETERMINATION OF MICROGLIAL-MEDIATED NEUROTOXICITY 2.9.1 Preparation and Treatment of Human Fetal Microglia Human fetal microglia were seeded into 24-well plates at 1.5 x 105 cells per well. Following 48 hr pre-incubation in serum-free medium, cells were treated with A p ^ 2 (5 uM), 4-AP alone, or APi_42 in combination with 2 m M 4-AP following 30 min preincubation with 4-AP, 4-AP alone and Ap42_i After 48 hours incubation with stimuli, cell free supernatants were transferred to primary rat hippocampal neurons plated on glass coverslips. Figure 2-4 illustrates the procedures carried out for the determination of microglial mediated neurotoxicity including control experiments which contained conditioned medium without microglial exposure. microglial conditioned medium Incubate with stimuli for 48 hrs m i c r o g l i a transfer Incubate with supernatant for 16 hrs n e u r o n s DAPI staining conditioned medium Incubate with stimuli for 48 hrs no m i c r o g l i a transfer Incubate with supernatant for 16 hrs n e u r o n s > DAPI staining Figure 2-4. Experimental procedure used to determine the effects of microglial conditioned medium on neuronal survival. Following incubation of primary human fetal microglia with stimuli for 48 hrs, microglial supernatant (microglial conditioned medium) was transferred to cultures of primary rat hippocampal neurons. Neurons were incubated with microglial conditioned medium for 16 hrs and subsequently stained with DAPI and visualized under a fluorescent microscope. A similar procedure was used to determine the effects of stimuli but lacking microglia (unconditioned medium); these experiments served as controls. 2.9.2 Isolation of Primary Rat Hippocampal Neurons Isolation of primary rat hippocampal neurons has been described previously (Sheldon et al., 2004a). . Briefly, 2 to 4 day-old Wistar rats were anaesthetized, decapitated and the hippocampi removed. The hippocampi were then enzymatically treated and mechanically dissociated and the resulting cell suspension was plated at a 5 2 density of 5-7 x 10 neurons cm" onto glass coverslips coated with poly-D-lysine and laminin. The initial growth medium was DMEM/F-12 supplemented with 10% fetal bovine serum (Invitrogen Canada, Burlington, Ont., Canada). After 24 hr, this medium was changed to serum-free Neurobasal Medium A (Invitrogen Canada). Cultures were then fed every 4-5 days by half-changing the existing medium with fresh Neurobasal Medium A . Glial proliferation was inhibited 48 hr after initial plating by adding 5-10 m M cytosine arabinoside (Sigma-Aldrich). Primary neurons were used 12-15 days after plating since at this time point cultured hippocampal neurons are more susceptible to toxicity (Sheldon et al., 2004b). Primary hippocampal neurons were treated with microglial conditioned medium or medium controls (conditioned medium not exposed to microglia) for 16 hrs. 2.9.3 Determination of Neuronal Damage from Microglial Conditioned Medium Following treatment, cells were fixed with 4% PFA in 0.1 M PBS. Following a wash in PBS, cells were incubated in DAPI (Molecular Probes) at 1 ug/mL in PBS to visualize nuclei. Cells were then washed in water and mounted onto glass slides using gelvatol, examined under a Zeiss light microscope and photographed using a cooled C C D camera. The percentage of damaged neurons was determined by counting the number of condensed or fragmented nuclear stained neurons by the overall number of positively DAPI stained neurons (at X200 magnification). Control experiments consisted of incubating neurons with conditioned medium without microglial exposure. Data are presented as mean ± S E M and significance determined by one-way A N O V A and Newmann-Keuls post-hoc multiple comparison test (p < 0.05). 2.10 IN VIVO STUDIES OF Api-42 MEDIATED MICROGLIAL ACTIVATION AND NEURONAL DAMAGE; EFFECTS OF 4-AP ADMINISTRATION 2.10.1 Injection of Ap 1.42 Into CA1 Region of Rat Hippocampus and Administration of 4-AP The procedure used for APi_42 injection in vivo is routinely used in our laboratory and has been published previously by other laboratories (Giovannini et al., 2002; Kowall et al., 1991; Weldon et a l , 1998). Briefly, male Sprague-Dawley rats (250-280g; Charles River, Canada) were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), and then mounted in a stereotaxic apparatus (Kopf Instruments, Tujunga, CA). A p ^ 2 or AP42-1 (1 nmol in 2 pi) was injected slowly (0.2 ul/min) into the granule cell layer of the dentate gyrus of the hippocampus (AP: -3.6 mm, M L : -1.8 mm, D V : -3.2 mm, according to the atlas of Paxinos and Watson, 1986) using a 10 uL Hamilton syringe. A diagram illustrating the site of injection of A p M 2 into the dentate gyrus of rat hippocampus is shown in Figure 2-5. 4-AP (Sigma, St. Louis, MO) was dissolved in 0.9% saline and administered i.p. at 1 mg/kg 15 minutes prior to AP1-42 injection followed by once-daily injections of 1 mg/kg for 7 days. The doses of 4-AP used in this study are based on previous studies using 4-AP in rats (Haroutunian et a l , 1985; Casamenti et al., 1982). A l l animal experiments were approved by the Animal Care Committee of the University of British Columbia. Figure 2-5. The Dentate Gyrus of the Rat Hippocampus. The syringe indicates on a coronal section of the hippocampus, the upper cell body layer of the dentate gyrus, where AP1-42 was injected. 2.10.2 Determination of APi^-Induced Neuronal Damage and Microglial Activation In Vivo Seven days post-APi_42 injection, anesthetized rats were transcardially perfused with heparinized cold saline followed by 4% PFA. Brains were postfixed overnight in the same fixative and then placed in 30% sucrose for cryoprotection. Serial coronal sections (40 urn) through the hippocampus were cut on a cryostat. For immunohistochemistry, free-floating brain sections were permeabilized with 0.2% Triton X-100 and 0.5% B S A in 0.1 M PBS for 30 min, blocked in PBS containing 0.5% B S A and 10% normal goat serum (NGS) for 30 min and then incubated overnight at room temperature in PBS containing 5% NGS and primary antibodies. The following primary antibodies were used: mouse anti-NeuN (1:1000, Chemicon, Temecula, CA) for neurons and mouse anti-EDl (1:500, Serotec, Oxford) for activated microglia. For controls, primary antibodies were omitted. The following day, sections were incubated with biotinylated secondary antibody (1:200; Vector Laboratories, Burlingame, CA) and visualized with A B C elite system (1:200; Vector Laboratories), and developed in 3,3'- diaminobenzidine (DAB) kit (Sigma). The number of NeuN- or EDI-positive cells in the superior blade of the dentate granule cell layer was conducted on three consecutive sections. Representative photomicographs were taken (at x200 magnification) and counting was performed using a Zeiss Axioplan 2 flourescent microscope (Zeiss) equipped with a D V C camera (Diagnostic Instruments) and Northern Eclipse software (Empix Imaging). A l l quantitative analyses were carried out in a blinded manner with values expressed as means ± S E M . 2.11 PEPTIDES AND REAGENTS 2.11.1 Preparation of APi^ 2 and AP42-1 The amyloid beta peptide (APi_42) and reverse peptide (AP42-1) were purchased from California Peptide (Napa, CA) and fresh stock solution prepared according to a method described previously (Walker et al., 2001) with slight modifications. Briefly, AP1.42 was prepared by first dissolving the peptide in 35% acetonitrile (purchased from Sigma, St. Louis, MO), diluted to 1.5 m M with sterile water, and then to 500 ^ M with incremental additions of PBS with vortexing in-between additions. The Ap solution was subsequently incubated at 37°C for 18 hrs to promote Ap fibrillization and aggregation and then stored at -20°C. The final working concentration used in experiments was 5 u M APi_42- A similar procedure was followed for preparation of reverse peptide AP42-1. The vehicle control was prepared as described for the preparation of APi_42 with omission of the peptide. Electron microscopy was used to confirm the aggregation of AP1.42 in solution. Briefly, the Ap sample (5 uL) was placed on Formvar-coated copper grids (Ted Pella Inc., Redding, CA) for 1 min. Excess solution was removed and then negatively stained with 2% acqeous uranyl acetate for 1 min, washed and then air dried. The grids were then examined on a JEM-100CX electron microscope (JEOL, Japan) at 80 kV. Figure 2- 6 is a representative photomicrograph of A) A P i ^ fibrils before heating and B) aggregated A p M 2 after heating at 37°C for 18 hrs. Before heating, immature fibrils appeared occasionally as a matrix-like meshwork however, after heating, these fibrils tended to appear as globular structures approximately 0.2-0.3 u M in width. Since the same procedure was used to dissolve the different lot numbers of AP peptide used, it was assumed that similar aggregation was achieved between the different batches of Ap peptide used in experiments. A D i r e c t H,.y . l Z O O O O x Figure 2-6. Representative photomicrographs of the AP1-42 preparation before and after heating. Negative staining results indicate that A) Ap fibrils exists before heating and that B) Ap aggregates form after incubation at 37°C for 18 hrs. 2.11.2 Reagents 4-aminopyridine (4-AP), guanosine-5'-0-(3-thiotriphosphate) (GTPyS), iberiotoxin, lanthanum (La 3 +), 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), SKF96365, and tetraethylammonium (TEA) were purchased from Sigma (St. Louis, MO) and apamin was a generous gift from Dr. Church, U B C . The FcyRII receptor antibody was purchased from Beckman-Coulter (Miami, FL) and C X C L 8 (IL-8) was purchased from Peprotech (Rocky Hi l l , NJ). 2.12 STATISTICAL METHODS Data are presented as mean ± S E M . Statistical comparisons were made using a Student's t-test to determine significant differences between control and treatment groups with significance set at p < 0.05. In cases where more than two groups were compared, a one-way A N O V A was used, followed by Neuman-Keuls post hoc multiple comparison test with significance set at p < 0.05. A l l statistical tests were carried out using GraphPad Prism 3.0 software. Chapter 3: EFFECTS OF Api.42 ON MEMBRANE POTENTIAL, MEMBRANE CURRENTS and FcyRII EXPRESSION IN HUMAN MICROGLIA In this chapter, the acute effects of AP1-42 on microglial membrane potential, membrane current expression and longer term effects on microglial expression of the FcyPvTI receptor are determined. The activation process in microglia has been associated with altered signaling which include membrane K + current expression (Norenberg et al., 1992; Fischer et al., 1995). Whole-cell electrophysiology was used to determine the acute effects of AP1.42 on membrane currents and membrane potential (V m ) . Since, FcyRII is a receptor coupled to a non-selective cationic channel and functional responses of microglia, the expression of FcyRII was also determined using an antibody binding study. 3.1 RATIONALE Microglial cells are capable of expressing ion channels that selectively conduct K + , H* or CI" (Eder, 1998). Other currents in microglia, such as inward N a + and voltage- gated L-type C a 2 + have occasionally been reported however, the presence of these currents in microglia is not common (Eder, 1998). Under resting conditions, microglia express an inward rectifier solely (Ilschner et al., 1995; Kettenmann et al., 1990; McLarnon et al., 1997). The upregulation of currents in microglia is dependent on several factors including the state of activation of microglia, the presence of stimuli and time of adherence to surfaces. Incubation with LPS, a potent activator of microglia, for periods of 12 hrs leads to the expression of a transient outward K + current in response to a depolarizing step in rodent microglia (Norenberg, 1992; Norenberg et al., 1994). This current was attributed to the upregulation of Kv l .3 channels (Norenberg et al., 1993). The induction of K + currents appears to be highly correlated with the activation state of these cells since under resting conditions in culture (ramified or ameboid morphology) microglia do not express outwardly rectifying K + currents but are activated in response to a stimulating agent. Another outward K + current can be induced in rodent microglia when GTP (guanosine 5'-0-(3-thiotriphosphate) is applied intracellularly through the patch pipette indicating that G proteins can regulate the upregulation of currents in microglia (Ilschner et al., 1995). The rapid expression of this current indicated that it was constitutively present in the cell membrane. A current with similar properties to the GTP-induced current was activated with external application of complement (Ilschner et al., 1996) and ATP (adenosine triphosphate) (Kettenmann et al., 1993; Walz et al., 1993). Human microglia also express a high conductance calcium activated K + channel (BK-Kc a ) sensitive to both V m (membrane potential) and [Ca ]i (intracellular calcium) (McLarnon etal., 1997). Rodent microglia express both proton and anion currents. The induction of proton currents is not correlated with the functional state of microglia (Klee et al., 1999) rather the currents are increased with cell swelling (Morihata et al., 2000). Proliferation, stretch or swelling of microglia is accompanied by activation of anion currents (Schlichter et al., 1996; Eder, 1998). A high conductance anion channel sensitive to V m has been reported in human microglia (McLarnon et al., 1997). As described in Section 1.4.3.1.1, numerous receptors have been described as mediating Ap actions in microglia. However, only a few of these receptors are coupled to signal transduction systems which could lead to alterations in membrane currents (i.e. a receptor complex (consisting of o^Pi integrin, CD47, CD36), FPR and CD36 alone. The Fey receptor in microglia has received recent attention since it is involved in microglial mediated phagocytosis of opsonized Ap which was thought to be an effective therapy in reducing levels of Ap in A D brain (Bard et al., 2000). The Fey receptor in particular is directly coupled to and activates a non-selective cationic channel in macrophages (Young et al., 1983a; 1983b) and activates signaling pathways in microglia which include phosphatidylinositol 3-kinase (PI-3K), extracellular signal regulated kinases (ERK) and mitogen-activated protein kinases (MAPK) activation (Song et al., 2004). The FcyRII class of receptors appeared to be the most highly expressed class in human microglia (Lue et al., 2002). Microglia in the brains of A D and non-demented individuals constitutively express all classes of Fey receptors: Fey RI, II, III, with expression being greater in A D than non-demented individuals (Akiyama and McGeer, 1990; Peressetal., 1993). The acute effects of APi_42 on human microglial membrane potential and current expression have been presented in this chapter. Since cell potential and membrane currents are important signaling events in the activation process of microglia, I carried out a series of whole-cell patch clamp experiments to determine the effects of Api-42 acutely on V m and outward currents and I determined the involvement of the Fcyll receptor in mediating A P M 2 effects on cell potential. 3.2 RESULTS 3.2.1 Effect of AP1.42 on Membrane Current Expression; Voltage Clamp Studies These experiments were designed to examine effects of full length APi^2 on membrane currents induced by depolarizing stimuli. Unless noted, microglia were held at a potential of -60 mV. A representative current response to a depolarizing step from a holding potential of -60 mV to + 20 mV under unstimulated conditions is shown in Figure 3-1. Currents elicited under unstimulated conditions in response to a depolarizing step were generally small (range 40 to 70 pA) and attributed to leak currents. This result is consistent with previous work on human (McLarnon et al., 1997; 1999) and studies on rodent (Ilschner et al., 1995; Kettenmann et al., 1990; Norenberg et al., 1994) microglia where outward K + currents in unstimulated microglia in response to a depolarizing step were generally absent. Within minutes of acute application of A p ^ 2 to human microglia, a rapidly activating, non-inactivating outward current was observed in response to a depolarizing step (Figure 3-1). To inhibit the contribution of any anionic currents present in microglia to the outward current, all membrane current experiments were performed with bath solution containing NPPB (500 pM). Overall, the amplitude of the current induced with acute application of AP1-42 in response to a depolarizing step in human microglia increased fourteen-fold (to 850 ±102 pA, n=7 cells) from control values (mean amplitude 59 ± 6 pA, n=7 cells). Experiments were designed to determine the specificity of the outward current. The analysis of tail currents (see below) indicated that the outward current was due to K + . +20 mV -60 mV J ~ L _ control 5 (iM Ap l J l 2 recontrol < I L _ 50 ms Figure 3-1. Typical current induced with acute application of Api_42 to human microglia In control, a small current representative of leak current was elicited with a depolarizing step to +20 mV from a V\{ of -60 mV. Following acute application of AP1-42 (5 uM), a large outward K + current was evident in response to a depolarizing step and wash-off of the peptide decreased the current to control level in response to a depolarizing step. The figure is a representative recording from one cell. 3.2.1.1 The APi-42-Induced Outward Current is Attributed to K* In order to determine the current's threshold for activation, a series of depolarizing steps in 10 mV increments was applied from -60 mV to a maximum depolarization of +20 mV. A typical family of I/V is presented in Figure 3-2. A plot of current amplitude versus step potential (I/V plot) indicated that the current was outwardly rectifying with a threshold of activation near -40 mV. Overall, the current's mean threshold for activation was determined to be -33.7 ± 2.4 mV (n = 4 cells). A +20 mV 50 ms V(mV) Figure 3-2. Voltage-dependent activation of Ap ̂ -induced outward current. A) Outward current, induced by A p l J t 2 (5 uM), with a series of depolarizing steps in 10 mV increments (applied from -60 mV to a maximum level of+20 mV). B) The I- V relationship constructed from the pulse protocol shown in panel A. The outward current induced by acute APi_42 was outwardly rectifying with a threshold of -40. mV. The figure is a representative recording from one cell. The reversal potential of the outward current was determined using a protocol consisting of a depolarizing step to +20 mV (applied from a holding potential of -60 mV) followed by varying the step potential from -100 to -20 mV (increments of 20 mV). The tail currents induced by varying the step potentials from (-100 to -20mV) are shown in Figure 3-3A. The arrows denote peak tail currents at the start of the different test potentials. These tail currents rapidly inactivated to baseline at the holding potential (Vh) of -60 mV. A typical plot of tail currents versus step potentials (-100 to -20mV) is shown in Figure 3-3B. Overall, the current's reversal potential was -76 ± 3.6 mV (n=6 cells) close to the equilibrium potential for K + (in this study EreV for K + = -84 mV). Therefore, these results indicate that the APi_42-induced K + current was outward and due t o K + . These results were confirmed since addition of the non-selective K + channel blocker 4-AP (2 mM) in the presence of Api.42 inhibited the current to 52% of control in response to a depolarizing step and the current in response to a depolarizing step recovered after wash-off of 4-AP (Figure 3-4). Overall, 4-AP at 2 and 5 m M in the maintained presence of APi_42 reduced the APi^-induced K + current in response to a depolarizing step to 58 ± 7.3% (n=3 cells) and 38 ± 6.6% (n=3 cells). Tetraethylammonium (TEA) (10 mM), a non-selective K + channel blocker, inhibited the outward K + current to 28 ± 5.9% (n=3 cells) of the Api^-induced current in response to a depolarizing step. Overall, the outward K + current elcited in response to a depolarizing step was induced by A p ^ 2 in 40% of cells and was present for periods of up to 10-15 minutes. -20 mV +40 mV -60 mV v. -40 mV -60 mV -80 mV B -100 mV -120 - - y j D ^ J o -60 -40 V(mV) -40 -60 ] 50pA| 50 ms Figure 3-3. Determination of the APi_42-induced outward current's reversal potential via analysis of tail currents. A depolarizing step from -60 mV to +20 mV was followed by a secondary step to potentials varying from -100 mV to -20 mV. The resulting tail currents elicited with the secondary steps from -100 mV to -20 mV in a representative experiment are shown in A. The arrows indicate the peak of the tail currents at the start of the different test potentials. A plot of tail current amplitudes versus step potential is shown in B. The current's reversal potential was determined as -78 mV which is close to the equilibrium potential for K + . +20 mV r -60mvJ control 50 ms Figure 3-4. The Api ̂ -induced outward K4" current is inhibited by the non-selective K* channel inhibitor 4-AP A representative recording from one cell is shown. The first trace is a typical current evoked in control solution with a depolarization step to +20 mV. AP1.42 (5 uM) elicited an outward K + current with the same step depolarization. Application of 4-AP (2 mM) with APi-42 reduced the outward JC current to 52% of control. The current recovered subsequent to wash off of 4-AP with APi_42 maintained in bath solution. 3.2.2 The Involvement of a G protein in the AP1-42 Induction of an outward K + current The possibility that a G protein may be involved in the induction of the outward K + current upregulated by AP1.42 in human microglia was tested since a previous study indicated that intracellular application of the non-hydrolyzable analogue of GTP, GTPyS, upregulated an outward K + current in macrophages (McKinney and Gallin, 1993). The outward K + current in macrophages had similar properties to the outward K + current induced by AP1.42 including rapid activation, non-inactivation during a depolarizing step and sensitivity to 4-AP. In the present study, GTPyS was added into the electrode solution and dialysed into the cell upon rupture of the seal in the whole-cell patch clamp mode. In these experiments, the extracellular bathing solution contained NPPB (500 uM) in order to inhibit any contribution of anion channels to outward currents induced by cell depolarization. Intracellular application of GTPyS induced a rapidly activating outward K + current in response to a depolarizing step within minutes of forming a whole-cell seal, however, the current showed rundown within ten minutes of its upregulation. A typical profile of the outward current's amplitude over time is presented in Figure 3-5. Rundown of the GTPyS-induced current may be due to equilibration of GTPyS between the electrode and cell. Overall, upon formation of a whole-cell seal, the mean current amplitude in response to a depolarizing step was 147 ± 49 pA (n=5 cells). In response to a depolarizing step, intracellular GTPyS induced an outward K + current in 70% of cells with an average peak current amplitude of 1149 ± 113 pA (n=9 cells). +20 mV [ -60 mV J upon seal formation intracellular GTPyS 4 min intracellular GTPyS intracellular GTPyS 7 min "50 ms Figure 3-5. Typical profile of the intracellular GTPyS-induced outward current. The top trace represents the voltage step (-60 mV to +20 mV) applied. The bottom four traces are currents at the times indicated after dialysis of GTPyS in response to a depolarizing step. Intracellular application of GTPyS (10 pM) via the electrode induced an outward current within minutes of rupture of the cell membrane in the whole cell patch clamp mode. A representative experiment indicates that the current amplitude increased transiently and randown within 10 minutes of seal formation. The current induced with intracellular GTPyS in response to a depolarizing step was similar in characteristics to the APi .42-induced K + current (Figure 3-6A,B). The GTPyS-induced K + current in response to a depolarizing step was rapidly activating, slow to plateau and did not inactivate with time typical of the A p i .42-induced K + current. The peak amplitude in response to a depolarizing step to + 20 mV from a holding potential of -60 mV of the GTPyS-induced K + current (1149 ± 113 pA; n=9 cells) was not significantly different from the amplitude of the A p i .42-induced K + current (850 ± 1 0 2 pA; n=7 cells) (p>0.05). Overall, the threshold for activation of the GTPyS- induced current was found to be -28 ± 1 . 7 mV (n=4 cells) which was not significantly different from the threshold for activation of the APi^-induced K + current (-33.7 ± 2.4 mV; n=4 cells) (Figure 3-6A; p > 0.05). Tail current measurements of the GTPyS- induced outward current indicated that the current had an overall reversal potential of -68 ± 1 . 1 mV (n=4) which was not significantly different from the reversal potential of the APi .42- induced outward K + current (E r e v = -76 ± 3 . 6 mV; n=6 cells) (Figure 3-6B; p > 0.05). The difference in reversal potentials of the GTPyS induced outward current (-68 mV) and the calculated reversal potential for K + in these experiments (-84 mV) may be due to contribution of a leak current to the overall whole-cell current induced with intracellular GTPyS. A representative result of the effect of 4-AP applied extracellularly on the GTPyS- induced K + current in response to a depolarizing step is shown in Figure 3-7. 4-AP (2 mM) reduced the GTPyS-induced K + current to 65% of control and the current recovered upon wash off of 4-AP similar to the 4-AP effect on the APi-42-induced K + current (Figure 3-7). Overall, 4-AP (2 mM) reduced the GTPyS-induced outward K + current to 61 ± 9.5 % of control (n=6 cells); a similar inhibitory effect 4-AP was observed on the APi_42-induced K + current in response to a depolarizing step (to 58 ± 7.3% (n=3 cells); p > 0.05). Since properties of the GTPyS-induced outward K + current were similar to the K + current induced with acute application of AP1.42 in response to a depolarizing step, the results would suggest that A P M 2 induces an outward K + current via activation of a G protein in human microglia. A - • - A P i ^ l(pA) - O - GTPyS B Figure 3-6. The current induced with intracellular GTPyS and the current induced with extracellular APi_42 had similar thresholds for activation and reversal potentials. A) The threshold for activation was determined as described above (Section 3.2.1.1). An I/V plot for the threshold of activation is shown from a representative experiment for each of GTPyS and APi-42-induced outward currents. Results indicate that the threshold for activation for each of the currents is similar: GTPyS -31 mV; APi_42 -38 mV. B) The reversal potential for each of the currents was determined as described above (Section 3.2.1.1). An I/V plot of tail currents from a representative experiment for each of the GTPyS and AP1-42 currents indicates that the reversal potentials are similar: GTPyS -70 mV; AP1-42 -78 mV. +20 mV -60 mV- GTPyS GTPyS + 2 mM 4-AP 2 | _ in< N 50 ms Figure 3-7. Effect of 4-AP (2 mM) on the intraceUular GTPyS-induced outward current Current evoked upon rupture of the cell membrane with GTPyS in the intracellular recording solution in response to a depolarizing step to +20 mV. The outward K + current elicited with intraceUular GTPyS for the same step depolarization. Extracellular application of 4-AP (2 mM) reduced the outward K*̂  current to 65% of the control. Wash-off of 4-AP allowed the current to recover. 3.2.2.1 Concentration-Dependent Inhibition of the GTPyS-induced Outward \C Current by 4-AP and Several Other K* Channel Inhibitors Since induction of an outward K + current was more common with intracellular application of GTPyS than with acute application of Api.42 and costs of peptide are high, I carried out further pharmacology on the outward K + current induced with intracellular GTPyS. Effects of 4-AP concentrations on the amplitude of the GTPyS-induced K + current were used to construct a concentration-response curve (Figure 3-8). A concentration dependent inhibition of the GTPyS-induced outward K + current with 4-AP was observed where C denotes control in the concentration-response plot. Overall, 4-AP was found to have an IC50 of 5 m M for inhibition of the outward K + current induced by intracellular application of GTPyS. T E A (10 mM) blocked the K + current induced with intracellular GTPyS to 23.8 ± 3.7% of control level (n=3 cells). The GTPyS-induced K + current was unaffected by the high conductance calcium-dependent K + channel (BK-type Kc a ) inhibitor iberiotoxin (50 nM) or by the small conductance calcium-dependent K + channel (SK-type Kc a ) inhibitor apamin (100 nM). These results indicate that upregulation of the APi-42-induced K + current is mediated through a G-protein and is not a B K - or SK-type K c a channel. A n outwardly rectifying current with a similar threshold for activation of -30 mV and sensitivity to 4-AP and T E A has been reported in rat astrocytes and referred to as a delayed rectifier type of K + current (KDR) (Bordey and Sontheimer, 1999). 1.0-, 100 [4-AP] mM Figure 3-8. Concentration-dependent inhibition of the intracellular GTPyS-induced outward K1" current by 4-AP. 4-AP was applied in the extracellular bath solution and amplitudes of the GTPyS- induced currents were measured in the presence of 4-AP and normalized to control amplitudes, C (current amplitude prior to 4-AP application). Results are a summary of: n=4 cells for 1 mM; n=6 cells for 2 mM; n=10 cells for 5 mM; n=4 cells for 10 mM and n=3 cells for 20 mM. 3.2.3 Molecular Biology Analysis of the Outward K* Current Based on electrophysiological data, the channel induced with acute Api_42 displayed the profile of a delayed rectifier type of K + channel. The expression of a series of K v channels ( K v l . l , 1.2, 1.3, 1.5, 1.6, 2.1, 3.1) with delayed rectifier K + channel properties reported previously in human cells (Jiang et al., 2002) were examined in unstimulated human microglia or cells incubated with A0i_42 for 10 min, 0.5, 1 and 2 hrs using RT-PCR. Results from a representative experiment are shown in Figure 3-9A. Results indicate a time-dependent increase in Kv3.1. A l l other K v channels showed basal expression in unstimulated microglia. A n abundant increase in the expression of Kv3.1 was observed after 10 min incubation with APi_42- The expression of Kv3.1 peaked at 0.5 hr then decreased at 1 and 2 hrs. No expression of Kv3.1 channel was evident at 4hrs APi.42 exposure. The absence of Kv3.1 expression under control conditions and transient expression with APi_42 incubation is in agreement with the transient upregulation of an outward K + current induced with acute A P M 2 in whole cell patch clamp experiments (Section 3.2.1). K v l . l , 1.2, 1.3 and 2.1 remained unchanged with treatment with Api.42 whereas Kv l .5 and K v l . 6 decreased in expression from control levels as the treatment time with AP1.42 increased. Incubation with AP42-1 (5 | J M ) at 10 min, 0.5, 1 and 2 hr did not alter Kv3.1 channel expression from control. Densitometry analysis of PCR product band intensities demonstrated the effect of Api.42 to time-dependently increase Kv3.1 channel expression with no effects of APi_42 to increase K v l . l , 1.2, 1.3, 1.5, 1.6 and 2.1 channel expression. Results are summarized in Figure 3-9B. AP1.42 significantly increased Kv3.1 channel expression at 10 min by 5.6 fold as compared to control (p < 0.05). Incubation with A p M 2 for 30 min significantly increased Kv3.1 channel expression further by 13 fold as compared to Kv3.1 channel expression in control (p < 0.001). Kv3.1 channel expression increased at 1 hr by 3 fold as compared to Kv3.1 channel expression in control; this increase was not significant (p > 0.05). Similarly, Kv3.1 channel expression was increased at 2 hr AP1.42 incubation by 3 fold relative to control, however the increase was not significant (p > 0.05). Furthermore, relative mRNA levels of K v l . l , 1.2, 1.3, 1.5, 1.6 and 2.1 channels induced with different AP1-42 treatment times were not significantly different from control (p > 0.05). A AP..42 Kv3.1 G3PDH B > < 4 z ¥ 3 1 1 • control • 10 min • 30 min A p ^ 2 • 1 h r A p , ^ • 2 h r A p ^ 2 Kv1.1 Kv1.2 Kv1.3 Kv1.5 Kv1.6 Kv2.1 Kv3.1 Figure 3-9. Effects of A|V-i: on Kv expression in human microglia A) A representative RT-PCR experiment of A P M 2 (5 uM) treatment for 10 min, 0.5, 1 and 2 hr on Kvl . l , 1.2, 1.3, 1.5, 1.6, 2.1 and 3.1 channel expression from n=3 independent experiments. G3PDH served as a reaction standard. B) Summary of relative mRNA levels of Kv channels induced by APi. 4 2 . Results are expressed as mean ± SEM from n=3 independent experiments. One-way ANOVA and Newman- Keuls multiple comparison test was used to evaluate statistical significance (̂ indicates statistical significance from control p < 0.05; ^indicates statistical significance from control p < 0.001). 3.2.4 Effect of Api^j on Membrane Potential of Human Microglia; Current Clamp Studies Overall, the average resting V m of human microglia was -35.6 ± 2.2 mV (n=16 cells). Within seconds of acute application, AP1.42 induced an immediate transient depolarization of microglia. A representative effect of Api-42 on V m is presented in Figure 3-10A. A p ^ 2 induced a transient change in V m (AV m ) (peak-baseline) of +8.5 mV with a 11/2 of 75 sec. ti/2 was determined as the time between the half-maximal points of the transient V m response. Overall, the A V m from baseline induced by acute AP1.42 was +8.9 ± 0.9 mV; tm of 73 ± 8 sec (n=5 cells). Acute application of AP42-1 had no effect on V m (n=3 cells; p > 0.05). A representative experiment indicating no effect of AP42-1 on membrane potential is shown in Figure 3-1 OB. It was of interest to compare the effects of Api.42 with effects of high K + solution on depolarization of human microglia. A high K + solution has been shown previously to induce depolarization of human microglia (McLarnon et al., 1999). A representative result with application of high extracellular K + (40 mM) to human microglia is presented in Figure 3-10C. High K + induced a transient depolarization with a A V m of +33.5 mV and ti/2 of 33 sec. Overall, high K + induced a transient depolarization with a A V m of +28.3 ± 6.2 mV and t i / 2 of 27 ± 7 sec (n=3 cells). Furthermore, application of either vehicle or bath solution alone did not induce a significant change in V m (n = 3 cells; p > 0.05). A representative trace indicating no effect of bath solution on membrane potential is shown in Figure 3-10D. B -20 -30 -40 -50 -10 -20 ^ -30 £ -40 -50 -60 -70 A042-1 0 20 40 60 80 100 120 time (sec) 20 40 60 80 100 120 time (sec) > E. E > 0 -10 > -20 B > -30 -40 -50 PSS 0 20 40 60 80 100 120 time (sec) Figure 3-10. Effects of A|3i^2, Ap 4 2-i, High K + and PSS on V m of Human Microglia Changes in membrane potential (Vm) were recorded with acute application of A) APi^ 2 (5 uM); B) Ap 4 2-i (5 u.M); C) 40 mM solution D) normal bath solution (PSS). 3.2.5 Effect of APi_42 on FcyRII Receptor Expression Since acute Api_42 induces depolarization of human microglia (Section 3.2.4) and reports indicate that the Fey receptor is linked to a non-selective cationic channel which induces cellular depolarization when activated, the effect of APt_42 on Fey receptor expression and of Fey receptor inhibition on A p i ^ 2 induced depolarization was investigated. After 30 min and 48 hr incubation of human microglia with A p ^ 2 (5 uM), optical density of cell surface FcyRII was determined using a monoclonal antibody of FcyRII. The effect of AP1-42 stimulation on FcyRII expression is presented in Figure 3-11. A P i ^ 2 significantly upregulated FcyRII expression for both treatment times: by 20% at 30 min (n=6; p<0.01) and by 36% at 48 hrs (n-6; p< 0.001). • 30 min • 48 hr Figure 3-11. APi^-induced expression of FcyRII The effect 30 min and 48 hrs A [51_42 treatment on FcyRII expression in human microglia was determined. The optical density of a monoclonal antibody to FcyRII after both 30 min and 48 hr treatment was measured and normalized to control. Results are a summary of n=4 independent experiments for 30 min treatment and n=6 independent experiments for 48 hr treatment. * indicates statistically significant from control (p<0.01); ** indicates statistically significant from control (p<0.001). Voltage clamp studies were also carried out to determine the effects of acute APi- 42 on inward currents in human microglia (personal communication with Dr. S. Jeong). Representative experiments of the effects of Api.42, AP42-1 and vehicle on inward currents in human microglia are shown in Figure 3-12. Application of vehicle had no effects on inward currents in human microglia (Figure 3-12A). Acute application of A P i ^ 2 (5 uM) led to an immediate transient inward current (Figure 3-12B) which corresponded with the depolarization observed previously (Figure 3-10A). I next investigated the effect of FcyRII inhibition on A p ^ 2 induced inward currents of human microglia. FcyRII receptors on microglia were inhibited by incubating microglia with FcyRII antibody (1:500) for lhr at 37°C prior to treatment as has been described previously (Mitrasinovic and Murphy, 2003). Inhibition of the FcyRII antibody for 1 hr prior to A p i ^ application, abrogated the induction of an inward current with acute Api_42 (Figure 3-12C). These results would indicate that the inward current induced with acute AP1.42 was mediated by activation of FcyRII. Acute application of AP42-1 (5 uM) had no effect on inward currents in human microglia (Figure 3-12D). The effect of A P i ^ 2 to induce an inward current through FcyRII activation was confirmed using current clamp studies (personal communication with Dr. S. Jeong). Representative experiments are shown in Figure 3-13. Acute application of AP1-42 (5 uM) led to an immediate transient depolarization (Figure 3-13A) as previously shown in Figure 3-1 OA followed by a small hyperpolarization indicative of K + channel activation. In a separate set of experiments, inhibition of FcyRII for 1 hr abrogated the effects of acute APi_42 (5 uM) to induce an immediate transient depolarization of human microglia (Figure 3-13B). These results indicate that binding of Api_42 to FcyRII leads to an inward current, a corresponding cellular depolarization and subsequent K + channel activation. A | Vehicle B j AP1-42 c j FcyRII Ab + Ap D J A342-1 |lOOpA 40s Figure 3-12. A(3î 2 induces an inward current in human microglia mediated by FcyRII activation. Representative traces of the effects of acute A) vehicle B) 5 uM AP1.42 C) 5 u.M Api. 42 in the presence of FcyRII inhibition D) 5 uM AP42.1 on inward currents of human microglia. C) Inhibition of FcyRII inhibition with FcyRII antibody incubation for 1 hr prior to acute APi.42 (5 uM) application inhibited the APi_42 induced inward current (B). A)Vehicle and D) AP42-1 had no effects on inward currents. -40 Vm -50- -60 J B -30-1 -40' Vm -50-1 -60 FcyRII Ab + A3^ 2 ^ ^ ^ ^ 40s Figure 3-13. The APi.42-induced transient depolarization of human microglia is mediated by FcyRII activation B) Inhibition of the FcyRII with an antibody for 1 hr prior to acute application of Api-42 (5 uM), prevented the transient depolarization induced with acute Api_42 (A). These results would indicate that the depolarization induced by acute APi_ 4 2 is mediated by FcyRII activation. 3.3 CONCLUSION Results from this section show that acute application of Api_42 induces a non- inactivating outward K + current via a G- protein. This current is sensitive to 4-AP and TEA. Methods of molecular biology indicate that the channel activated by A0i_42 is the delayed rectifier type Kv3.1. Results also indicate that acute application of AP1.42 results in transient depolarization of microglia mediated via activation of Fcyll receptors. Under unstimulated conditions, rodent (Eder, 1998; Ilschner et al., 1995; Kettenmann et al., 1990) and human (McLarnon et al., 1999) microglia lack an outward K + current. This is the first study reporting the upregulation of a rapidly activating, non- inactivating, outwardly rectifying K + current in human microglia in response to acute AP1.42 (Figure 3-1). This current differs from the transiently inactivating K + current attributed to activation of the "n-type" K + channel (Kvl.3) with LPS in rodent microglia (Norenberg et al., 1992; 1993). Expression of Kv l .3 requires cell exposure to LPS for periods in excess of 12 hrs. Effects of Ap on membrane currents have been reported previously; however, treatments with Ap were long-term and carried out using rat microglia (Chung et al., 2001). Incubation with AP25-35 or AP1.42 for 12-24 hours led to a hyperpolarization of cells attributed to upregulation of a K v current (Kvl.3 and Kvl .5) . Upregulation of K + channels in microglia are considered an indicator of cellular activation (Fischer et al., 1995) and may counteract the depolarizing effects of certain metabolites present during inflammation (Norenberg et al., 1992; Illes et al., 1996). However, the electrophysiological results in this study indicate that upregulation of a K + channel in response to acute Api_42 may serve as an early signaling event in the activation process of human microglia by the peptide. Another novel finding from our patch clamp studies was that the intracellular application of the non-hydrolyzable GTP analogue, GTPyS, induced an outward K + current in human microglia (Figure 3-5) similar to the outward K + current upregulated by acute application of AP1-42 (Figure 3-1). The GTPyS activated outward K + current displayed similar kinetics of activation (Figure 3-6A), ion selectivity (Figure 3-6B) and sensitivity to 4-AP (Figure 3-7) and T E A as the APi_42-hiduced outward K + current which indicated that AP1.42 induces an outward K + current via activation of a G protein. Previous studies have indicated that Ap mediates its effects on microglia by activating a pertussis toxin sensitive G protein, G; (Lorton et al., 1997; Tiffany et al., 2001) as well as other second messengers (Combs et al., 1999).. G protein-induced K + currents have been reported previously in other cell types including murine macrophages (McKinney and Gallin, 1992), T lymphocytes (Schumann and Gardner, 1989) and mast cells (McCloskey and Cahalan, 1990). Interestingly, the K + current induced by intracellular application of GTPyS in macrophages (McKinney and Gallin, 1992) was also sensitive to pertussis toxin and 4-AP but unaffected by both apamin, an inhibitor of small conductance calcium activated K + channels, and charybdotoxin, an inhibitor of large and intermediate conductance Ca2+-activated K + channels similar to the GTPyS-induced K + current observed in this study in human microglia. These results could suggest a common G protein signaling pathway in the induction of an outward K + current in both human microglia and murine macrophages. In this study, data from the kinetics of activation and rectification indicated that the channel was a delayed rectifier type of K + channel similar to an LPS-induced delayed rectifier K + channel reported previously in rat astrocytes (Bordey and Sontheimer, 1999). The transient expression of Kv3.1 channels in response to short incubations with APi_42 (10 min to 0.5 hr) (Figure 3-9) is in agreement with the results from patch clamp studies indicating upregulation of an outward K + current within minutes of acute APi_42 application followed by its rundown within 10-15 minutes (Figure 3-1). Pharmacological characterization of the Ap ̂ - induced outward K + current indicated sensitivity to both 4- A P (2 mM) (Figure 3-4) and to T E A (10 mM). Previous work has demonstrated Kv3.1 sensitivity to 4-AP and T E A (Grissmer et al., 1994). Previous reports have also indicated that Kv3.1 mRNA is regulated by basic fibroblast growth factor, depolarization, Ca and cAMP levels (Perney et al., 1992; Gan et al., 1996) and also that increased expression of Kv3.1 was observed in a subset of proliferating T lymphocytes (Kv3.1 referred to as Type / K + channel in T lymphocytes; Grissmer et al., 1992) in autoimmune diseases including experimental allergic encephalomyelitis (EAE) (Chandy et al., 1990) and arthritis (Grissmer et al., 1990). These reports support the involvement of second messengers such as a G protein in coupling A p ^ 2 to an outward K + current and of the Kv3.1 channel in mediating this current in human microglia. Furthermore, the immediate depolarization and progressive increase in [Ca ]i with acute APi_42 application in this study could act as precedents for the rapid induction of Kv3.1 mRNA observed in microglia. It is also possible that the rapid induction in Kv3.1 expression within minutes of incubation with Api.42 and the upregulation of the outward K + current observed within minutes of acute application of A(3M2 in whole-cell patch clamp experiments are independent events in microglia. The earliest time point reported for Kv channel protein translation to occur was 15 minutes (Deutsch, 2002) which is a longer time point than that required for the outward current to be induced upon acute application of AP1.42 (within 3-4 minutes) in whole-cell patch clamp experiments. The induction of the outward K + current upon acute application of Api_42 within minutes would indicate that the channel protein is either rapidly inserted into the plasma membrane from intracellular vesicles or that the channel is constitutively located in the plasma membrane and is non- functional but requires phosphorylation by second messengers to become activated. The rapid transcription of Kv3.1 could be a compensatory mechanism to replenish the intracellular Kv3.1 protein pool needed for induction of an outward K + current induced with acute Api_42- Molecular biology studies indicated basal expression in human microglia of all delayed rectifiers ( K v l . l , 1.2, 1.3, 1.5, 1.6, 2.1) with the exception of Kv3.1. Treatment with AP1.42 did not alter levels of K v l . l , 1.2,1.3, 2.1 whereas Kv l .5 and 1.6 decreased as the incubation period with AP1-42 increased. No corresponding delayed rectifier currents, i.e. transient inactivating K + current typical of Kv l .3 upregulation, were observed in unstimulated conditions nor after Api.42 application. This discrepancy between the presence of K v mRNA under unstimulated and stimulated conditions and the lack of corresponding current has been reported previously in microglia (Norenberg et al., 1993; Khanna et al., 2001). This discrepancy was attributed to constitutive transcription and possible stimulus control of post-translational synthesis of the channel protein, translocation or insertion of the channel protein into the plasma membrane. In our study, microglia expressed mRNA for Kv l .5 and 1.6, however, expression decreased as Api_42 incubation time increased. Previous reports have also reported a decrease in Kv l .5 current expression as microglia transform from a resting to activated state and proliferate (Kotechaand Schlichter, 1999). Results from this study also indicate that the mechanism through which Api-42 induces depolarization in microglia (Figure 3-10) is via activation of the FcyRII since AP1-42 time-dependently increased FcyRII expression (Figure 3-11) and inhibition of FcyRII completely abolished the depolarization induced with AP1-42 (Figure 3-13). This was confirmed since acute application of A P M 2 induced an inward current which was inhibited by incubation of microglia with an FcyRII antibody (Figure 3-12). The mean resting membrane potential of microglia in our study was approximately -36 mV which agrees with previous reports of resting membrane potentials in rodent microglia (Chung et al., 1998; 2001). This immediate depolarization induced by acute A p M 2 through the FcyRII receptor could in turn activate Kv3.1 since the immediate transient depolarization induced with acute application of AP1.42 led to an immediate small hyperpolarization of microglia (Figure 3-13). Kv3.1 has been shown previously to be regulated by cellular depolarization (Perney et al., 1992). A role of FcyRII receptor activation in the upregulation of Kv3.1 is supported from previous studies indicating that FcyRII activation leads to pertussis toxin sensitive G protein activation and subsequent inflammatory mediator production in macrophages (DuBourdieu and Morgan, 1990; Bronner et al., 1990). This is also the first study to report the acute action of Api.42 to induce a transient depolarization of microglia. Chapter 4: MODULATION OF Ap^-INDUCED INTRACELLULAR SIGNALING AND FUNCTIONAL RESPONSES OF MICROGLIA BY 4-AMINOPYRIDINE: IN VITRO AND IN VIVO 4.1 RATIONALE The molecular mechanisms by which microglia become activated and then mediate inflammatory responses in the CNS remain elusive. The activation process in microglia has been associated with specific cell signaling pathways and factors including membrane K + current expression (Norenberg et al., 1992; Fischer et al., 1995), altered calcium homeostasis (Vehratsky et al., 1998), activation of p38 M A P kinase (McDonald et al., 1998; Pyo et al., 1998) and activation of the transcription factor N F K B (Combs et al., 2001; Bonaiuto et al., 1997). K + channel activation has been shown to have wide spread effects on microglial cell function including proliferation (Schlichter et al., 1996; Kotecha and Schlichter, 1999), migration (Schilling et al., 2004) and respiratory burst activity (Khanna et al., 2001). Modulation of the intracellular signaling pathways associated with microglial activation in response to inflammatory stimuli such as Ap could serve as a rational approach to inhibit inflammation in A D . 4-aminopyridine (4-AP) has been used as a potential therapeutic in A D due to its ability to prolong the nerve action potential, enhance calcium influx and consequently increasing neurotransmitter release (Glover, 1982). A D pathophysiology is associated with deficits in neurotransmitter systems including the cholinergic, noradrenergic, serotonergic, dopaminegic, GABAergic and glutamatergic systems (Nordberg, 1992) and 4-AP has been reported to increase levels of these neurotransmitters in the brain (Tapia and Sitges, 1982; Dolezal and Tucek, 1983; Hu and Fredholm, 1991; Boireau et a l , 1991; Scheer and Lavoie, 1991; Tapia et al., 1985; Tibbs et al., 1989). 4-AP also possesses favourable properties as an A D therapeutic as compared to other non-selective potassium channel inhibitors such as tetraethylammonium (TEA) since 4-AP has a rapid onset of action and the capability to cross the blood brain barrier (Soni and Kam, 1982). Results from two clinical trials using 4-AP as a putative potentiator of cognitive function in patients with A D have been published (Wesseling et al., 1984; Davidson et al., 1988). One study (Wesseling et al., 1984) reported reduced mental deterioration in patients receiving 4-AP whereas a subsequent study (Davidson et al., 1988) reported no significant difference between patients receiving 4-AP and those receiving placebo. These discrepancies as well as the differences in study design between the two trials precluded a direct comparison and it was concluded that 4-AP warranted further evaluation as a potential A D therapeutic (Wiseman and Jarvik, 1991). As results from Chapter 3 indicate, 4-AP inhibits the upregulation of an outward K + current induced by acute APi_42. Molecular biology analysis indicated that the underlying channel is likely Kv3.1. Thus, I have investigated further the potential of 4-AP as a modulator of APi-42-induced intracellular signaling pathways and functional responses of human microglia. In vitro, I have studied the effects of AP1-42 on intracellular calcium [Ca2 +]j and modulation by 4-AP of APi_42-induced [Ca2 +]j signaling, p38 M A P K and N F K B activation, the expression and production of pro-inflammatory mediators and potential neurotoxicity in vitro. In vivo, I have investigated the effects of 4-AP on A p ^ 2 - induced microglial activation and neurotoxicity. The results from this study indicate that 4-AP exhibits wide spectrum anti-inflammatory activity both in vitro and in vivo. 4.2 RESULTS 4.2.1 Effect of APi^»2 on IntraceUular Calcium of Human Microglia; Inhibition by 4-aminopyridine 4.2.1.1 Effects of Apx^2 on [Ca2+]j Calcium spectrofluorometry was used to investigate the effects of AP1-42 and of 4- A P on APi -42-induced intracellular calcium responses in human microglia. In these experiments, levels of [Ca2 +]j are specified as ratios of 340/380. Baseline calcium levels ranged from 0.2 to 0.3. In standard physiological solution (PSS), AP1-42 (5 uM) acutely applied induced a slow progressive increase in [Ca 2 +];, to a plateau; a representative result is shown in Figure 4.-1. A peak increase in [Ca 2 +]i of 0.07 was attained within 380 sec (n = 39 cells) (Figure 4-1). Overall, the mean amplitude of the response was 0.11 ± 0.01 (n = 98 cells). As shown in Figure 4-1, the replacement of Ca 2 +-PSS with A P U 2 in C a 2 + - free-PSS following the plateau inhibited this increase by 96%. Overall, in Ca2+-free PSS the APi .42-induced response was decreased by 93 ± 5 % (n=98 cells). Washout of AP1.42 with Ca 2 +-PSS had no effect to alter the plateau level of [Ca2+]i induced by AP1-42 indicative that the responses induced by Api.42 were not reversible. Furthermore, acute application of A p 4 2 - i (5 uM) (n= 68 cells) or vehicle (n=83 cells) had no effect on [Ca2 +];. JO 0.15 - 0.1 - 0.05 - 0 I i i i 1 1 i 1 0 100 200 300 400 500 600 700 time (sec) Figure 4-1. Acute application of APi^u induces a slow, progressive increase in [Ca2+]j Representative trace of the increase in [Ca2+]j induced by Api.42 (5 uM) (n=21 cells). Subsequent application of C a 2 + free PSS with Api.42 maintained in solution decreased [Ca2+]j to baseline levels. The decrease in [Ca ]i induced with AP1.42 in Ca -free solution suggests that an influx pathway mediates the increase in [Ca ]j with A(i 1-42- This point was further tested with application of AP1-42 in Ca2+-free PSS. As shown in Figure 4-2 (n= 23 cells), APi_42 induced a negligible transient increase in [Ca2 +]j with a peak amplitude of 0.03 and a ti/2 of 185 sec. Overall, in C a 2 + free PSS, AP1.42 induced a mean increase in [Ca 2 + ] jof0 .02± 0.01 and mean tm of 170 + 15 sec (n=38 cells). These results indicate that APi_42 induces an increase in [Ca2 +]j primarily through a C a 2 + influx pathway. 0.6 0.5- 0.4 • o £ 0.3 H. CO 0.2 • 0.1 - 0 Ca 2 + -PSS 100 200 Ca2 +-free PSS 300 time (sec) 400 500 —i 600 Figure 4-2. In Ca -free solution, Api.42 induces a small increase in [Ca ]i. The standard PSS solution was first exchanged for Ca2+-free PSS. A negligible increase in [Ca ]j was induced by acute application of Api-42 (5 uM) in Ca -free PSS (n=23 cells). This representative trace indicates that the increase in [Ca2+]j induced with APi_42 is primarily due to influx of Ca . Previous work from this laboratory has reported that microglia do not possess voltage-gated C a 2 + channels (McLarnon et al., 1999) and that the major C a 2 + influx pathway in human microglia is store-operated channel (SOC). I investigated the possibility that SOC could mediate the entry of C a 2 + induced, by AP1.42 by using SKF96365, an agent which inhibits SOC in human microglia (Choi et al., 2003) and in other cells (Li et al., 1999). Upon attainment of a plateau in [Ca 2 +]i with AP1-42, application of SKF96365 (50 ^ M ) did not alter [Ca2 +]j (Figure 4-3; n=23 cells). The subsequent exchange of bath solution to Ca -free PSS caused an immediate decrease of [Ca 2 +]i to baseline levels. Overall, SKF96365, applied at the peak of the response, did not significantly alter the increase in [Ca ]j induced by acute AP1.42 (n=81 cells). This result would indicate that SOC does not contribute to the influx pathway induced by acute A P i ^2 in human microglia. 0.15 • 0.1 - 0.05 • 0 I i i i i i i i i i 0 100 200 300 400 500 600 700 800 900 time (sec) Figure 4-3. SKF96365, an inhibitor of SOC, had no effect on the increase in [Ca2+]j. A representative trace of the effect of SKF96365 on the increase in [Ca2+]j induced by APi_42 (n=21 cells). Subsequent to the plateau in [Ca2+]j induced by acute APi_4 2 (5 u M ) in C a 2 + PSS, application of SKF96365 (50 u M ) had no effect to alter the increase in [Ca2+]j induced by APi_4 2 . Application of C a 2 + free PSS reduced [Ca2+]j to baseline. This result would indicate that the C a 2 + influx pathway induced by A P i . 2+ 42 was not due to SOG. Note that wash-out of APi_42 did not reduce [Ca ] i . 4.2.1.2 Effects of 4-AP on Api^2-induced Ca influx pathway A representative figure (Figure 4-4) demonstrates the effects of 4-AP on the APi_ 42-induced [Ca2 +]j increase. APi_42 induced a progressive increase in [Ca 2 +]i with an amplitude of 0.06 (mean, n=26 cells). Application of 4-AP (2 mM) near the peak of the response decreased [Ca 2 +]i to baseline levels (Figure 4-4). The effect of 4-AP to decrease [Ca 2 +]i was rapid. Overall, 4-AP inhibited the APi_42-induced increase in [Ca2 +]j by 96 ± 0.53 % (n = 149 cells). These results may indicate that cellular depolarization mediated by 4-AP inhibition of voltage-dependent K + channels inhibits influx of C a 2 + induced by acute APi-42- As shown in Figure 4-5, L a 3 + (50 uM), an inhibitor of C a 2 + permeable channels, applied at the peak of the AP1-42 response inhibited completely the APi .42-induced C a 2 + increase (n=12 cells). Overall, L a 3 + inhibited the APi^-induced increase in [Ca 2 +]i to baseline levels (n=44 cells). These results would indicate that APi_42 induces C a 2 + influx through a cationic permeable channel which is sensitive to depolarization by agents such as 4-AP. Ca 2 + PSS 0.4 i 0.35 • 0.3 0.25 • 0.15 0.1 • 0.05 • 0 0 100 200 300 400 500 600 700 800 time (s) Figure 4-4. The non-selective K1" channel inhibitor, 4-AP, inhibits the APi_42-induced increase in [Ca2+]j. A representative trace of the effect of 4-AP on the C a 2 + influx pathway induced by APi_42 (n=26 cells). Subsequent to the slow progressive increase in [Ca2+]i induced by acute APi_4 2 (5 u M ) in Ca PSS, application of 4-AP (2 m M ) rapidly decreased [Ca2 +]i to baseline levels. This result would indicate that the Ca 2 + influx pathway induced by APi_4 2 is sensitive to depolarization induced by 4-AP. 0.15 • 0.1 • 0.05 - 0 -I —i 1 1 1 1 1 1 1 0 100 200 300 400 500 600 700 800 time (sec) Figure 4-5. La , an inhibitor of Ca permeable channels, inhibits the A p - - induced increase in [Ca2+]j. A representative trace of the effect of L a 3 + on the C a 2 + influx pathway induced by APi_42 (n=12 cells). Subsequent to the slow progressive increase in [Ca2+]j induced by acute A p ^ (5 uM) in Ca 2 + PSS, application of L a 3 + (50 uM) decreased [Ca2+]j to baseline levels. This result would indicate that the C a 2 + influx pathway induced by AP1-42 is mediated through a cationic channel. 4.2.2 Effects of Api^2 on p38 MAP Kinase and N F K B Activation in Human Microglia; Attenuation by 4-aminopyridine Effects of 4-AP on APi-42-induced p38 MAP kinase activation in human microglia p38 M A P kinase and N F K B activation have been implicated in linking of inflammatory stimuli to functional responses of microglia. Stimuli include LPS and AP which induce secretion of pro-inflammatory factors (Lee et al., 2000; McDonald et al., 1998; Combs et al., 2001; Bonaiuto et a l , 1997). Increased N F K B and p38 M A P K are observed in A D brain (Terai et al., 1996; Hensley et al., 1999). I have investigated the effects of 4-AP on p38 M A P kinsase and N F K B using immunocytochemical procedures (see methods section 2.4.1). The effects of 4-AP on Api_42-induced p38 M A P kinase activation in human microglia are presented in Figure 4-6A. Under basal conditions, low numbers of positively stained cells for phospho-p38 M A P kinase were detected. Stimulation with APi_42 (5 u M for 30 min) induced an increased expression of phospho-p38 M A P kinase in microglia (green), which was blocked if 4-AP (2 mM) was included with AP1-42 treatment. 4-AP itself showed no effect to alter phospho-p38 M A P kinase from control. Overall, p38 M A P kinase activation was analyzed from n=4 independent experiments. The results (Figure 4-6A, B) indicate that A P i ^ significantly increases (by 371%) the number of cells expressing activated p38 M A P kinase in human microglia (p < 0.001). 4- A P (2 mM), in the maintained presence of Api.42, resulted in a significant reduction (by 58%) in the number of cells expressing phospho-p38 M A P kinase as compared to stimulation with Api_4 2 alone (p < 0.001). 4-AP (2 mM) and Ap 42-i (5 uM) did not significantly alter the basal level of phospho-p38 M A P kinase stained cells (p > 0.05). Control A P 1 - 4 2 A P ^ 2 + 4-AP 4-AP Phosphop38 + DAPI +4-AP Figure 4-6. Effects of 4-AP on APi .42-induced p38 MAP kinase activation A) Representative photomicrographs of phosphorylated p38 (phospho-p38) stained microglia. The green and blue indicate staining for phospho-p38 and DAPI positive nuclei, respectively. Under control conditions, little or no phospho-p38 expression was observed. A P i _ 4 2 (5 uM) treatment of microglia for 30 min induced an intense expression of phospho-p38. A p 1.42 in the maintained presence of 4-AP (2 mM) inhibited expression of phospho-p38. Application of 4-AP (2 mM) alone had no effect on phospho-p38 expression. B) The percentage of phospho-p38 positive microglia relative to total cells are shown. Data are means ± SEM from four independent experiments. * indicates significance compared with control p < 0.001 and ^indicates significance compared with Ap 142 p < 0.001. Scale bar = 50 um. In positive control experiments, LPS stimulation of microglia (for 30 min) resulted in a significant increase in p38 M A P K positive microglia (to 73.7 ± 5.8%) from the number of p38 M A P K positive cells in untreated conditions (control: 3.7 ± 1 . 3 % ; p < 0.001). Effects of 4-AP on Afij-42-induced NF-kB activation in human microglia The modulatory actions of 4-AP were also investigated on APi-42-induced activation of the transcription factor, N F - K B . The presence of the active subunit of N F - K B , p65, was determined using immunocytochemistry. As shown in Figure 4-7A, a low number of cells express p65 under basal conditions (green). Stimulation with A P — (5 \\M for 8 hr) increased the expression of p65 in microglia which was blocked i f 4-AP (2mM) was included with A P M 2 treatment. 4-AP itself showed no effect to alter p65 levels from control. Overall, p65 levels were analyzed from n=5 independent experiments. The results (Figure 4-7A, B) indicate that A P — induces a significant increase (by 493%) in p65 expressing cells (p < 0.01). 4-AP in the maintained presence of A P — significantly decreased (by 60%) the number of cells expressing p65 (p < 0.01). 4-AP (2 mM) alone induced an increase in p65 positive cells, however, the increase was not significant (p > 0.05). AP42-1 did not alter basal levels of p65 expressing cells (p > 0.05). In positive control experiments, LPS stimulation of microglia (12 hrs) resulted in a significant increase in p65 positive microglia (to 27 ± 2.9%) from the number of p65 positive cells in untreated conditions (control: 0.3 ± 0.1%; p < 0.001). A Control AP 1 - 4 2 A P 1 - 4 2 + 4-AP 4-AP p65 + DAPI + 4-AP Figure 4-7. Effects of 4-AP on APi.42-induced N F K B activation A) Representative photomicrographs of p65 (the active subunit of N F K B ) stained microglia. The green and blue indicate staining for p65 and DAPI positive nuclei, respectively. Under control conditions, little or no p65 expression was observed. A(ii_42 (5 uM) treatment of microglia for 8 hrs induced an intense expression of p65. Ap— in the maintained presence of 4-AP (2 mM) inhibited expression of p65. Application of 4-AP (2 mM) alone had no effect on p65 expression. B) The percentage of p65 positive microglia relative to total cells are shown. Data are means ± SEM from five independent experiments, îndicates significance compared with control and **indicates significance compared with APi_42; p < 0.01. Scale bar = 50 fim 4.2.3 Effects of A P — on Expression of Pro-Inflammatory Mediators; Attenuation by 4-aminopyridine Microglia are a major source of pro-inflammatory mediators. In this study, I examined the effects of 4-AP on A p i .42-induced expression of the pro-inflammatory cytokines (IL-lp, IL-6, TNF-a), chemokine (CXCL8 (IL-8)) and inducible enzyme COX-2. Results from a representative experiment are shown in Figure 4-8A. Overall, microglia express C X C L 8 (IL-8) constitutively under unstimulated conditions, whereas IL- lp , IL-6, TNF-a and COX-2 were not expressed. After 8 hr incubation with 5 u M A p — , increased expression of all pro-inflammatory mediators was observed. In the presence of A p — , 4-AP (2 mM) decreased the expression of all pro-inflammatory mediators. 4-AP alone had little or no effect on expressions of pro-inflammatory mediators. AP42-1 (5 uM) was ineffective to alter expression of pro-inflammatory mediators from control. Densitometry analysis of PCR product band intensities showed that 4-AP reduces APi .42-induced pro-inflammatory mediator expression. A summary of relative mRNA levels of inflammatory mediators induced with AP1-42, 4-AP and of A P — + 4-AP is shown in Figure 4-8B (n=7 experiments). Overall, A P — (5 uM) significantly increased relative mRNA levels of all pro-inflammatory mediators (* indicates p < 0.05). A P — , in the presence of 4-AP led to a significant decrease in pro-inflammatory mediator expression relative to A P — stimulated levels (** indicates p < 0.05). Stimulation with 4-AP alone led to a small increase in levels of pro-inflammatory mediators relative to control, however, the increases were not significant (p > 0.05). Fold increases in relative pro-inflammatory mediator mRNA induced by AP1-42, 4-AP, A p — + 4-AP, and AP42-1 compared to control are summarized in Table 4-1. Overall, APi_42 increased the expression of pro-inflammatory mediators (relative to control) by (x fold increase): IL- lp : 3.8; IL-6: 6.8; C X C L 8 (IL-8): 1.8; TNF-a: 3.6; COX-2: 2.9. 4-AP alone increased the expression of pro-inflammatory mediators (relative to control) by (x fold increase): IL- lp : 1.3; IL-6: 1.5; C X C L 8 (IL-8): 1.1; TNF-a: 1.5; COX-2: 1.5. Inhibition of A p ^ 2 - induced expression of pro-inflammatory mediators by 4-AP was (x fold decrease): IL-1 P: 0.56; IL-6: 0.60; C X C L 8 (IL-8): 0.65; TNF-a: 0.61; COX-2: 0.51. A p 4 2 - i did not alter relative mRNA levels of pro-inflammatory mediators from control (p > 0.05). B ) > OH E i « "5 2.5 2 1.5 * 1 1 0.5 ] II u IL-1 (3 • control 0 A p ^ 2 + 4-AP • 4-AP I IL-6 IL-8 TNFKX COX-2 Figure 4-8. Effects of 4-AP on Ap—-induced expression of pro-inflammatory mediators Effects of A P — and of A P — in the presence of 4-AP on the expression of pro- inflammatory mediators by human microglia using semiquantitative RT-PCR. Expression of (A) TNF-a, IL-6, IL-lp, CXCL8 (IL-8) and COX-2 were examined in microglia incubated for 8 hrs with AP—, 4-AP, Ap — in the combined presence of 4-AP, or with medium alone. Stimulation of microglia with Ap 4 2 1 (5 uM) alone served as a control experiment. The results shown are a representative of seven independent experiments. The expression of G3PDH served as a reaction standard. B) Summary of relative mRNA levels of inflammatory mediators induced by A P — , 4-AP and combined A p — and 4-AP. Results are expressed as mean ± SEM from n=7 independent experiments. One-way ANOVA and Newman-Keuls multiple comparison post-test was performed to evaluate statistical significance (p < 0.05) ('indicates statistically significant from control; ** indicates statistically significant from A P — stimulated levels). Table 4-1. Fold increases in relative pro-inflammatory mediator mRNA induced by APi_42> 4-AP, AP1.42 + 4-AP, and AP42-1 compared to relative mRNA in control using semiquantitative RT-PCR A p W 2 Api.42 + 4-AP 4-AP Ap 4 2 . , IL-lp 3.8* 2.0 1.3 1.2 IL-6 6.8** 4.2** 1.5 1.4 CXCL8 (IL-8) 1.8* 1.2 1.1 1.1 TNF-a 3.6* 2.2* 1.5 1.4 COX-2 2.9* 1.5 1.5 1.3 * p< 0.05; **p<0.01 4.2.4 Effects of A P i ^ 2 on Production of Pro-Inflammatory Mediators; Attenuation by 4-aminopyridine I next investigated the effects of APi_42 exposure on microglial production of inflammatory factors and actions of 4-AP to modulate the effects of Ap on production of these inflammatory factors. The production of TNF-a, IL-6, IL-1 B and C X C L 8 (IL-8) were investigated after 48 hrs stimulation with AP1.42 in the presence and absence of 4- AP (2 mM) using ELISA. 48 hrs was chosen as the optimum time point to determine the modulatory actions of 4-AP on cytokine production using ELISA since protein levels continued to accumulate through 48 hrs and incubations with A P M 2 for periods longer than 24hrs could induce both direct and indirect effects of the peptide (Walker et al., 2001). A summary of results is presented in Figure 4-9. APi_42 increased secretion of TNF-a (by 2.3 fold; n=4 independent experiments) (Figure 4-9A); IL-6 (by 46 fold; n=3 independent experiments) (Figure 4-9B); IL- ip (by 1.9 fold; n=6 independent experiments) (Figure 4-9C); C X C L 8 (IL-8) (by 4.5 fold; n=4 independent experiments) (Figure 4-9D) and compared to basal levels in human microglia; all increases were significant (p < 0.001). AP1-42 in the maintained presence of 4-AP (2 mM) decreased levels of TNF-a (by 46%); IL-6 (by 73%); IL- lp (by 26%); C X C L 8 (IL-8) (by 47%) as compared to A p M 2 alone; all decreases were significant (p < 0.001). The changes in levels of pro- inflammatory mediators induced with 4-AP and AP42_i each alone were not significantly different from basal levels (p > 0.05). A summary of fold increases in pro-inflammatory mediator production induced by Api_42, 4-AP and A P M 2 in the present of 4-AP as compared to levels in control is presented in Table 4-2. A B + 2mM4-AP 200 2mM 4-AP Figure 4-9. Effects of 4-AP on APi_42-induced pro-inflammatory mediator production. Effects of AP1.42, 4-AP and AP1.42 in the maintained presence of 4-AP on pro- inflammatory cytokine secretion by human microglia using ELISA. Data are mean ± SEM of four independent experiments for (A) TNF-a; three independent experiments for (B) IL-6; six independent experiments for (C) IL-ip and four independent experiments for (D) CXCL8 (IL-8), each performed in duplicate. Human microglia were exposed to either medium alone, APi^,2 (5 uM), 4-AP (2 mM), AP1.42 in the presence of 4-AP or AP42-1 for 48 hrs. One-way ANOVA and Newman-Keuls multiple comparison post-test was performed to evaluate statistical significance (p < 0.001) ('indicates statistical significance from control; ^indicates statistical significance from Api_ 4 2 stimulated levels). Table 4-2. Fold increases in pro-inflammatory mediator production induced by Apt. 42,4-AP, APi_42 + 4-AP and AP42-icompared to levels in control APi.42 4-AP A p ^ 2 + 4 - A P AP42-1 I L - l p I 9*** 0.90 1.4** 1.0 IL-6 2.9 13** 1.2 CXCL8 (IL-8) 0.95 2.4 1.4 TNF-a 2 3*** 0.48 1.2 0.86 *p < 0.05; **p < 0.01; ***p < 0.001 The production of COX-2 was determined using immunocytochemistry and representative results are presented in Figure 4-1 OA. Stimulation with A P — (5 u M for 24 hrs) induced an increase in the number of COX-2 positive microglia (green) from control which was blocked with 4-AP (2 mM) in the maintained presence of A p i _42. 4- A P and Ap 4 2 - i each alone had no effect to alter COX-2 levels from control. Overall, results of the effects of A P — and of A P — on COX-2 expression were determined from n=6 independent experiments. A p — significantly increased the percentage of microglia expressing COX-2 by 5.1 fold from control levels (p < 0.001) (Fig. 4B). 4-AP (2 mM) in the maintained presence of A P — significantly decreased (by 0.43 fold) the percentage of COX-2 expressing cells (p < 0.01). 4-AP (2 mM) and A p 4 2 - i (5 uM) each alone induced an increase in the percentage of COX-2 positive microglia compared to unstimulated conditions, however, the increases were not significant (p > 0.05). In positive control experiments, stimulation of microglia with LPS (for 12 hr) resulted in a significant increase (by 54 fold) in COX-2 positive cells from untreated conditions (n-3 independent experiments; p < 0.001). A Control A p ^ 2 A | 3 1 J 4 2 + 4-AP COX-2 (green) + DAPI (blue) + 4-AP Figure 4-10. Effects of 4-AP on APi_42-induced COX-2 expressing microglia A) Representative photomicrographs of COX-2 positively stained microglia. The green and blue indicate staining for COX-2 and DAPI positive nuclei, respectively. Under control conditions, little or no COX-2 expression was evident. Treatment of microglia for 24 hrs with Afi i_42 (5 uM) induced an intense expression of COX-2. Ap11_42 in the presence of 4-AP treatment inhibited production of COX-2. 4-AP alone had no effect on basal levels of COX-2 production. B) The percentage of COX-2 positive microglia relative to total cells are shown under the different experimental conditions. Data are means ± SEM from six independent experiments. * indicates significance compared to control (p < 0.001) and **denotes significance compared to Api-42 (p < 0.01). Scale bar = 50 nm. 4.2.5 Effects of A P — on microglial mediated primary hippocampal neuronal toxicity; inhibition by 4-aminiopyridine An important study in my research program was to investigate A P — stimulated microglia as a possible source of neurotoxicity both in the presence and absence of 4-AP. The strategy used to investigate microglial mediated neurotoxicity has been described in Chapter 2 (Section 2.9) and is presented in Figure 4-11 A . Briefly, the supernatant of human microglia stimulated with Ap—, in the presence and absence of 4-AP (2 mM), for 48 hr (conditioned medium) was transferred to primary rat hippocampal neuronal cultures. Following 16 hr incubation with microglial conditioned media, neurons were stained with DAPI and the number of neurons with condensed nuclei were counted. Controls consisted of stimulating neurons with medium lacking microglia (unconditioned medium). As shown in Figure 4-1 IB, incubation of neurons with medium from A p — stimulated microglia resulted in increased numbers of neurons with condensed nuclei (bright fluorescent nuclei) and 4-AP (2 mM) in the maintained presence of A P — decreased microglial mediated neurotoxicity. 4-AP had no effect to alter basal levels of neuronal death or with AP42-1. Stimuli alone (unconditioned medium) had no effect on neuronal viability. Overall, neuronal toxicity from microglial conditioned medium and unconditioned medium was obtained from n=5 independent experiments. The percentage neurotoxicity induced by microglial conditioned medium (from microglia stimulated for 48 hrs with AP— , 4-AP, AP42-1, and A P — in the presence of 4-AP) as well as with unconditioned medium have been summarized in Figure 4-11C. Incubation of neurons with the supernatant of Api^2-stimulated microglia induced a significant increase (by 232%) in the number of neurons with condensed nuclei as compared to levels induced by unstimulated microglia (p < 0.001). 4-AP significantly reduced the amount of APi.42- induced microglial neurotoxicity by 54% (p < 0.001). Conditioned medium from microglia treated with 4-AP and AP42-1 each separately increased the level of neuronal death by 9% and 14% respectively as compared to basal levels, however, the increases were not significant (p > 0.05). Treatment of neurons with medium from unstimulated microglia did not alter the basal level of neuronal damage (p > 0.05). Incubation of neurons for 16 hrs with unconditioned medium containing AP1-42 (5 uM) alone and in the presence of 4-AP (2 mM), 4-AP alone and AP42-1 (5 uM) did not alter the basal percentage of neuronal damage (p > 0.05). medium Incubate with stimuli for 48 hrs or control microglial teg&g ^©^^ conditioned V ^ r ^ w r ? ? * : microglia transfer supernatant Incubate with supernatant for 16 hrs neurons • DAPI stainine Incubate with stimuli for 48 hrs Incubate with supernatant for 16 hrs unconditioned medium B transfer supernatant -> DAPI stainine no microglia control neurons A p ^ 2 + 4-AP DAPI + microglial conditioned medium 100 90 </> 80 c o L_ 70 3 a> c 60 T3 d) 50 O) ca E 40 CO T3 30 ~s 20 10 0 • unconditioned medium • microglial conditioned medium control ** Ap 1 - U + 2mM 4-AP Ap 4 2 . , 2m M 4-AP Figure 4-11. Effects of microglial conditioned medium from microglia stimulated with A |i 1.42, 4-AP each alone or in combination on neuronal survival. A) Strategy used to investigate microglial mediated neurotoxicity (taken from Figure 2-4) B) Representative photomicrographs of neurons treated with microglial conditioned medium. Condensed and fragmeneted nuclei were considered damaged neurons. Scale bar = 50 um C) Summary of microglial mediated neurotoxicity results from n=5 independent experiments. * indicates statistically significant from medium of unstimulated microglia (p<0.001); ** indicates statistically significant from conditioned medium of Ap 1.42 stimulated microglia (p<0.001). 4.2.6 In vivo effect of 4-aminopyridine on Ap—'induced neurotoxicity and microglial activation A n important aspect of my work was to determine the anti-inflammatory potential and neuroprotective actions of 4-AP in vivo. This was done by microinjection of A p — (1 nmol) into the dentate gyrus of rat hippocampus and at 7 days post-injection, both microglial activation and neuronal toxicity was determined (refer to Methods section 2.10). The effects of 4-AP were investigated in rats administered 4-AP (1 mg/kg) i.p. daily for 7 days. 4.2.6.1 4-AP reduces AP—'induced neurotoxicity in vivo I examined the effect of 4-AP on APi^-induced neuronal toxicity in vivo using NeuN, a marker of viable neurons. Representative NeuN positive staining results from Api.42 injection into the dentate granule cell layer of rat hippocampus, in the absence and presence of 1 mg/kg 4-AP, are presented in Figure 4-12A-D. Loss of NeuN positive granule neurons was evident at 7 days post A P — injection (Figure 4-12B) compared to vehicle (Figure 4-12A). As shown in Figure 4-12C, 4-AP was effective in protecting neurons from APi .42-induced damage. Little or no loss of neurons was observed with 4-AP alone (Figure 4-12D) or with AP42-1 alone. Overall, at 7 days after AP1-42 injection, the number of NeuN positive neurons in the superior blade of dentate granule cell layer decreased by 18% compared to vehicle (Figure 4-12E; p < 0.05). Treatment with 4-AP reduced the neurotoxic effect of A P — since the number of NeuN positive cells was significantly increased by 16 % in A p — injected brain administered 4-AP relative to the number of NeuN positive cells in peptide-injected brain (p < 0.05). No significant neuronal loss was observed in AP42-1- injected or 4-AP treated rat brain (Figure 4-12E; p>0.05). A) Vehicle B) A p 1 j | 2 C)Ap^ 2+4 -AP D) 4-AP NeuN Vehicle A P 1 - 4 2 A P ^ 2 + 1 mg/kg AP 4 2.., 1 mg/kg 4-AP Figure 4-12. Effects of 4-AP on A(} 142-induccd hippocampal neuron degeneration in vivo Representative photographs of tissue sections stained with NeuN antibody from superior blade of dentate granule cell layer taken from A) vehicle injected rats and rats treated with B) A p M 2 (1 nmol), C) A p M 2 plus 4-AP (1 mg/kg i.p.) and (D) 4-AP seven days after injection. (E) Quantification of the effects of AP1-42 and of AP1.42 in the presence of 4-AP on NeuN positive neurons. Data are mean ± SEM (n = 4/group). *p<0.05 vs control; **p<0.05 vs A p i ^ . Scale bar = 50 um. 4.2.6.2 4-AP reduces APi_42-induced microglial activation in vivo I next examined the effect of 4-AP on Api_42-induced microglial responses in vivo using EDI , a marker of microglial activation. Representative EDI positive staining results from Api_42 injection into the dentate granule cell layer of rat hippocampus, in the absence and presence of 1 mg/kg 4-AP, are presented in Figure 4-13A-D. Numbers of EDI positive microglia (Figure 4-13B) were considerably increased with peptide relative to vehicle (Figure 4-13A). 4-AP treatment attenuated the number of activated microglia in AP1-42 injected rat brain (Fig. 4-13C). Injection of 4-AP alone had no effect on numbers of activated microglia (Fig. 4-13D). AP42-1 had a small effect to increase the numbers of EDI positive cells (Fig. 4-13E). Overall, in APi^-hijected brain, the numbers of microglia were significantly increased by 18-fold compared to vehicle (Fig. 4-13F; p < 0.05). In APi^-injected brain, administration of 4-AP resulted in a significant reduction in the number of EDI positive microglia (by 68%) relative to numbers of microglia with AP1-42 (Fig. 4-13F; p < 0.05). No significant increase in the number of EDI positive microglia was found in 4-AP treated brain compared to vehicle (Fig. 4-13F; p > 0.05). A p 4 2 - i injection resulted in increased EDI positive microglia compared to vehicle, however, the increase was not significant (Fig. 4-13F; p > 0.05). The above results in vivo indicate that 4-AP is neuroprotective in APi^-injected brain and reduces the number of activated microglia induced by AP1.42. Figure 4-13. In Vivo effects of 4-AP on APi_42-induced microglial activation Representative photographs of tissue sections stained with EDI from superior blade of dentate granule cell layer taken from A) vehicle injected rats and rats treated with (B) A p M 2 (1 nmol) (C) A p ^ 2 plus 4-AP (1 mg/kg), (D) 4-AP or E) Ap 4 2-i (1 nmol) at seven days post-injection. F) Quantification of the effects of APi_4 2, 4-AP and of Api_4 2 in the presence of 4-AP on EDI positive microglia. Data are mean ± SEM (n = 4/group). *p< 0.05 vs control. ** p < 0.05 vs A p ^ . Scale bar = 50 fim 4.3 CONCLUSION My results show, 4-AP in both in vivo and in vitro assays, modulates AP1.42- induced microglial activation and subsequent functional responses including neuronal toxicity. Administration of 4-AP reduced AP1-42 mediated neurotoxicity and microglial activation in vivo (Figure 4-12; 4-13). In vitro, the results suggest that a possible mechanism through which 4-AP reduces neuronal toxicity is via effects on AP1-42- stimulated microglia (Figure 4-11). Acute application of APi_42 induced an increase in intracellular calcium [Ca 2 +]i through a C a 2 + influx pathway which was inhibited by 4-AP (Figure 4-4). In longer-term studies, 4-AP also inhibited APi^-induced activation of p38 M A P kinase (Figure 4-6) and N F K B activation (Figure 4-7) in human microglia. Furthermore, 4-AP inhibited APi^-induced functional responses of human microglia including the expression (Figure 4-8) and production of pro-inflammatory cytokines (IL- ip , IL-6, T N F - a ) , the chemokine C X C L 8 (IL-8) (Figure 4-9) and inducible enzyme COX-2 (Figure 4-10). A summary of the effects of 4-AP on AP1-42 induced intracellular signaling and functional responses in human microglia is shown in Figure 4-14. Na2+/C * In Vivo secretion f TNF-a IL-ip IL-6 1 • 1 I IL-8 * ! others •k • neurotoxicity microglia indicates sites of A P M 2 induced signaling and functional responses in microglia as well as in vivo which are modulated by cellular depolarization mediated by 4-AP blockade of an outward K + channel neuron Figure 4-14. Summary of the effects of 4-AP on APi^-induced intracellular signaling and functional responses in human microglia in this study. Administration of 4-AP markedly decreased APi^-induced hippocampal neuronal cell death in vivo (Figure 4-12). To my knowledge this study is the first to report therapeutic effects of 4-AP towards APi-42-mduced neurotoxicity in vivo. My results also show significant effects of 4-AP to reduce microgliosis in peptide-injected rat brain (Figure 4-13). The in vitro neuroprotection study (Figure 4-11) provided a clear indication that the presence of microglia is essential for APi^-induced neurotoxicity since Api.42 directly did not induce killing of neurons. Rather, the addition of conditioned medium from Ap ̂ -stimulated microglia induced significant killing of neurons (Figure 4-11) and further, the neuroprotective effects of 4-AP were clearly induced by the inhibition of APi^-stimulated microglia. These results support the findings that AP1.42 alone is not neurotoxic but rather that microglia may play a role in inflammation-mediated neurodegeneration in A D (McDonald et al., 1997; Minghetti et al., 1998). Specifically, microglial activation has been associated with neurodegeneration through the production of neurotoxic factors such as pro-inflammatory cytokines, NO, superoxide anion (McDonald et al., 1997; Griffin et al., 1998; Combs et al., 1999, 2000) and an unidentified neurotoxin (Giulian et al., 1995). In this study, AP1-42 caused microglial activation both in vivo (Figure 4-13) and in vitro (Figure 4-1; 4-6; 4-7) and also caused a significant increase in the production of pro-inflammatory mediators (Figure 4-9; 4-10); 4-AP inhibited these stimulatory effects of APi_42 in human microglia. These results would suggest that 4-AP is exerting its neuroprotective effects by deactivating microglia and subsequently inhibiting Api_42-induced production of pro- inflammatory mediators (Figure 4-9; 4-10) and other potentially unknown neurotoxic factors thereby reducing neurotoxicity (Figure 4-11). Ca ions play a central role in several cellular functions including regulation of cell volume, cell motility and serves as a signal transduction element to activate other membrane ion channels (e.g., Ca2+-activated K + and CI" channels) and transporters. The inhibitory effects of 4-AP on the Ap M 2 - induced C a 2 + influx pathway indicates that this influx pathway is sensitive to changes in membrane potential (Figure 4-4). Time frames for acute effects of APi_4 2 on membrane currents (Chapter 3) and intracellular C a 2 + should not be taken as written. The sequence of events occurring in these studies upon binding of A p M 2 to a receptor, likely FcyRTI, is depolarization, subsequent activation of a K + channel, likely Kv3.1 (as described in Chapter 3) which in turn increases the driving force for C a 2 + influx leading to the subsequent slow progressive increase in [Ca2 +]j. Inhibition of this K + channel with 4-AP reduces this driving force for C a 2 + to enter the cell leading to a subsequent decrease in [Ca2 +]j. The major influx pathway in microglia is store-operated (SOC) since microglia do not possess voltage-gated C a 2 + channels (McLarnon et al., 1999). Since the SOC inhibitor SKF96365 had no effect on the A p M 2 - induced increase in [Ca2 +]j, these results would indicate that the C a 2 + influx pathway induced by AP1-42 in human microglia was not SOC mediated. The inhibition of the Api_ 42-induced increase in [Ca 2 +]i by L a 3 + would suggest that the Api_4 2 effect was mediated through a non-specific cation permeable channel however, the specific identity of the pathway remains unknown. The Ca increase could be mediated by FcyRTI receptor activation since the FcyRTI receptor is coupled to a non-selective cationic channel which would allow Ca into the cell once activated. A P has been shown to activate a G protein-coupled extracellular calcium-sensing receptor (CaR) in neurons which stimulates a Ca2+-permeable, non-selective cation channel leading to increasing [Ca2 +]j (Ye et al., 1997). A CaR has been identified in rat microglia (Chattopadhyay et al., 1999) and thus, AP1.42 may be acting on a CaR in human microglia resulting in an increase in [Ca 2 +]i. Interestingly, a similar unidentified C a 2 + influx pathway to that induced with APi_42 in this study has been reported previously in human microglia in response to acute IFN-y (Franciosi et al., 2002). La had a similar slow inhibitory effect on the increase in [Ca ]i with acute IFN-y as it did on the [Ca ]j increase with acute A P M 2 reported in this study. The immediate inhibition of the A P i .42-induced C a 2 + entry pathway by 4-AP (Figure 4-4) indicates that membrane depolarization inhibits this influx pathway. It could also indicate that 4-AP is exerting effects other than non-selectively blocking K + channels such as inhibiting non-specific cation permeable channels or activating C a 2 + extrusion mechanisms such as the P M C A . In contrast to our findings, 4-AP has been shown to increase [Ca ]j in other cells including astrocytes, (Grimaldi et al., 2001) and monocytes 9+ (Lajdova et al., 2004) which would indicate that the effect of 4-AP on [Ca ]j differs between cell types studied. Ap-induced increases in [Ca ]; have been reported previously in microglia (Silei et al., 1999; Korotzer et al., 1995). 4-AP also inhibited AP1-42 activation of the SER-kinase, p38 M A P kinase, in human microglia (Figure 4-6). Ap has been shown previously to induce pro- inflammatory effects in microglia via activation of p38 M A P kinase (McDonald et al., 1998; Pyo et al., 1998; K i m et al., 2004) and inhibition of p38 M A P kinase resulted in decreased microglial inflammatory reactions and subsequent neurodegeneration (Giovannini et al., 2002) similar to the results of this study. The activation of p38 M A P kinase leads to the phosphorylation of a multitude of downstream kinases and transcription factors, regulates mRNA stability of pro-inflammatory cytokines such as T N F - a , and influences chromatin accessibility to transcription factors such as N F - K B (Koistinaho and Koistinaho, 2002). Therefore, altered p38 M A P kinase activation may provoke altered signaling pathways downstream of the kinase. Previous work has shown that delayed rectifier K + currents are inhibited by p38 M A P kinase in microglia and attributed this inhibition by p38 M A P kinase as the mechanism by which delayed rectifier currents are time-limited (Schilling and Eder, 2003). Therefore, the increase in phosphorylated p38 M A P kinase observed with A P — may play a role in the downregulating the outward K + current induced with A P — in human microglia (Chapter 3) and a similar time course of p38 M A P kinase activation by A P — (10-30 minutes) and downregulation of the outward K + current (Chapter 3) was observed in this study (within 10-15 minutes). In a separate study, inhibition of stress-induced K + channel activity by 4-AP in a myelocytic leukemic cell line resulted in decreased p38 M A P K phosphorylation (Gao et al., 2004). It was suggested that inhibition of K + channel activity by 4-AP resulted in decreased cell volume and subsequent inhibition of M A P K translocation at the centrosome, the major microtubule organization center of the cell. This reduction in K + channel activity, alteration in cytoskeletal proteins and subsequent inhibition of p38 M A P kinase translocation may be the mechanism through which 4-AP inhibits p38 M A P kinase activation in human microglia. The inhibition of the A p - - induced increase in phosphorylated p38 M A P kinase by 4-AP may also be due to the reduction by 4-AP in [Ca ]; levels induced by A P — , however, studies have indicated that calcium is not a requirement for phosphorylation of p38 but rather plays a role downstream of M A P kinases in mediating pro-inflammatory mediator release (Hide et al., 2000). The transcription factor N F - K B is one of the key molecules in the process of microglial activation on which several signaling pathways elicited by pro-inflammatory stimuli, converge (O'Neill and Kaltschmidt, 1997). N F - K B has been implicated in the transcription of numerous genes in microglia including the pro-inflammatory factors expressed by A P i ^ 2 stimulated microglia in this study: T N F - a , IL- ip , IL-6 (Combs et al., 2001; Kang et al., 2001; Heyen et al., 2000), the chemokine C X C L 8 (IL-8) (Ehrlich et al., 1998) and COX-2 (Egger et al., 2003). In unstimulated cells, N F - K B exists in a latent form complexed with an inhibitory protein of the IKB family. Upon activation, IKB proteins are targeted for degradation, thus allowing the translocation of the active subunit of N F - K B (p65/p50) to the nucleus and the transcription of target genes. Our results indicate that Api_4 2 increases the percentage of microglia expressing the p65 subunit of N F K B and that 4-AP inhibits this effect of APi_4 2 on N F K B (Figure 4-7). APi_4 2 has been shown previously to activate N F K B in microglia (Bonaiuto et al., 1997). This is the first study demonstrating the modulatory effects of 4-AP on N F - K B activation. It has been shown previously that N F K B activation is involved in the upregulation of an outward K + current in microglia since addition of a selective N F - K B inhibitor in the presence of a viral protein resulted in complete inhibition of the outward K + current (Visentin et al., 2001). A possible mechanism for 4^AP mediated inhibition of APi_42-induced N F - K B activation is through the reduction in the upstream signaling factor p38 M A P K (Figure 4- 6) since p38 M A P K may control N F - K B transactivation (Vanden Berghe et al., 1998; Madrid et al., 2001). As expected, the inhibitory effects of 4-AP on Api_42-induced N F - K B activation had subsequent inhibitory effects downstream to N F - K B in the AP1.42 signaling pathway in human microglia. The APi-42-hiduced gene expression of pro-inflammatory cytokines (IL- lp, IL-6, TNF-a), chemokine C X C L 8 (IL-8) and inducible enzyme COX-2 (Figure 4-8) and production (Figure 4-9; 4-10) of these factors were inhibited by 4-AP. AP1.42 has been shown previously to stimulate the expression of pro-inflammatory mediators in human microglia (Lue et al. , 2001a). A previous report indicated that 4-AP inhibited LPS-induced production of IL- ip in rat microglia (Caggiano and Kraig, 1998). The inhibitory effect of 4-AP was attributed to the inability of the cell to compensate the LPS- induced cellular depolarization in the presence of 4-AP. The neuroprotective properties of 4-AP in vitro (Figure 4-11) are consistent with actions to inhibit APi_42-induced expression and production of pro-inflammatory mediators by microglia. Since 4-AP was also able to reduce production of pro-inflammatory cytokines, C X C L 8 (IL-8) and COX-2 (Figure 4-9; 10), it is possible that 4-AP blocks other microglial secretory products involved in neurodegeneration. Interestingly, our results in vivo indicated that the administration of 1.0 mg/kg 4- A P was neuroprotective in AP1-42 injected rat brain (Figure 4-12). In a previous study examining the effects of 4-AP on memory performance in rats, lower concentrations of 4- A P (0.10-1.0 mg/kg) led to enhanced performance (Haroutunian et al., 1985). Administration of 1 mg/kg 4-AP is estimated to give a cerebrospinal fluid concentration of ~2-20 uM, falling to ~1 u M after 10-30 minutes (Lemeignan et al., 1984; Jankowska et al., 1982). The concentration of 4-AP which was effective in vitro in blocking microglial-induced intracellular signaling and functional responses including neurotoxicity in this study was in the m M range (-1000 fold higher than that used in vivo). This discrepancy between the concentration used in vitro and dose administered in vivo is due to the narrow therapeutic window of 4-AP. The maximum tolerable CSF concentration in humans is ~5 uM (Felts and Smith, 1994). The difficulty in administering higher concentrations of 4-AP in vivo is due its side effects since numerous types of K + channels are present in the CNS, each with differing sensitivities to 4-AP. The inhibition of microglial mediated neurotoxicity by 4-AP would indicate that the K + channel Kv3.1 induced by AP1.42 in human microglia (Chapter 3) is involved in mediating microglial neurotoxicity. K + channels in microglia are believed to participate in K + homeostasis and may play a role in cell proliferation/cell differentiation. In particular, 4-AP was shown to inhibit microglial proliferation (Kotecha and Schlichter, 1999). 4-AP non-selectively inhibits different voltage-gated K + channels directly regulating membrane potential and cell volume. But 4-AP may also act indirectly by affecting ion transport through other channels, exchangers, and pumps. Our results demonstrate that K + channels, [Ca2 +]j, p38 M A P K and N F - K B are pivotal signaling proteins/molecules to transduce A P M 2 actions in microglia. As the results from this study indicate, 4-AP not only inhibits the K + channel induced by Api_4 2 (Chapter 3) but as a consequence of this change in membrane potential, alters the phosphorylation of kinases and factors involved in gene transcription, subsequent gene expression and production of inflammatory products in human microglia. These results would indicate that these signaling molecules are somewhat integrated with each other. Moreover, the reciprocal cross-amplification or inhibition between mediators also implies the physiological significance of these signaling factors in the progress of inflammation. Our principal result in this work is the finding that block of a K + channel (likely Kv3.1) is a putative strategy to reduce Ap-induced inflammation. The present data suggests some degree of clinical efficacy for 4-AP in A D since the agent is permeable to the B B B . However, a major problem in the use of this agent clinically is that it has non- selective actions on other K + channels which can cause unwanted side effects. Development of a compound with specificity to block Kv3.1 could serve as a rational strategy to reduce inflammation and slow the progression of the pathology in A D . Chapter 5: CXCL8 (IL-8) POTENTIATION OF Ap—-INDUCED FUNCTIONAL RESPONSES OF HUMAN MICROGLIA 5.1 RATIONALE The chemokine C X C L 8 (IL-8) plays an important role in inflammation. C X C L 8 (IL-8) is an autocrine agent for microglia inducing chemotaxis of these cells to sites of injury (Cross and Woodroofe, 1999). This chemokine is also released by microglia (Ehrlich et al., 1998) activated by stimuli such as Ap (Nagai et al., 2001). Importantly, elevated levels of C X C L 8 (IL-8) (Galimberti et al., 2003) and C X C L 8 (IL-8) receptors (Xia et al., 1997) have been detected in A D brain. In a detailed analysis of gene expression profiling of A p — stimulated post-mortem adult human microglia isolated from non-demented individuals, C X C L 8 (IL-8) was observed to be strongly upregulated (Walker et al., 2001). These results suggest that C X C L 8 (IL-8) may play a role in the network of inflammatory responses which contribute to the pathogenesis of A D . In the present study, I have investigated the effects of C X C L 8 (IL-8) on A P — - induced expression of the pro-inflammatory cytokines TNF-a, IL-1 P, IL-6, the inflammatory enzyme COX-2 and the anti-inflammatory cytokines IL-10, TGFPi . In addition, in cases where expression were altered, I then examined C X C L 8 (IL-8) as a modulator of A P — actions to alter production of the agents. 5.2 RESULTS 5.2.1 Effect of A|3M2, CXCL8 (IL-8) Alone and Combined on Morphology of Human Microglia Morphologically, unstimulated microglia are typically ramified indicative of a resting state (Ling and Wong, 1993) (Figure 5-1 A). Stimulation with C X C L 8 (IL-8) for 24 hrs had little or no effect on cell morphology as indicated in Figure 5-IB with a majority of cells ramified in morphology. After 24 hrs incubation with aggregated APi_42 (5 uM) (Figure 5-1C), microglia showed an ameboid (activated) phenotype characterized by attenuated processes, swelling and presence of intracellular vacuoles (Walker et al., 2001). A similar activated morphology was observed after 24 hrs incubation with APi_42 and C X C L 8 (IL-8) (100 ng/mL) combined (Figure 5-1D). Morphology of microglia stimulated for 24 hrs with reverse peptide AP42-1 (5 uM) was similar to that of unstimulated microglia (Figure 5-1E). Addition of AP42-1 in combination with C X C L 8 (IL-8) had little or no effect to alter the ramified morphology of microglia (Figure 5-IF) as was observed with C X C L 8 (IL-8) stimulation alone (Figure 5-IB). A B c Figure 5-1. Effects of Api-42, CXCL8 (IL-8) each alone or in combination on morphology of human microglia Phase contrast images of human microglia after 24 hrs in (A) unstimulated conditions (B) CXCL8 (IL-8) (100 ng/mL) (C) A p M 2 (5 |iM) (D) A p M 2 and CXCL8 (IL-8) combined (E) Ap 4 2 - i and (F) Ap 4 2 . , and CXCL8 (IL-8) combined. (xlOO magnification; scale bar = 50 uM in A-F). 5.2.2 Effects of CXCL8 (IL-8) on Ap ^-Induced Expression of Pro-inflammatory Mediators I next investigated the expression of pro- and anti-inflammatory cytokines, C X C L 8 (IL-8) and COX-2 in microglia stimulated for 8 hrs with A p M 2 or C X C L 8 (IL-8) separately or both in combination. A single time point of 8 hrs was chosen for RT-PCR analysis since at this time point, optimal expression of inflammatory mediators was induced by A P i ^ 2 in human microglia. Representative results with APi_4 2 , C X C L 8 (IL-8) and combined A P M 2 and C X C L 8 (IL-8) on inflammatory mediator expression are shown in Figure 5-2. Unstimulated human microglia did not express IL- ip , IL-6, TNF-a, or COX-2 constitutively, however, C X C L 8 (IL-8) was expressed under basal conditions (Figure 5-2A). Stimulation with A P i ^ , 2 or C X C L 8 (IL-8) for 8 hrs induced the expression of IL- lp , IL-6, C X C L 8 (IL-8), TNF-a and COX-2. Incubation of human microglia with A$i^2 in the presence of C X C L 8 (IL-8) led to enhanced expression of all pro-inflammatory factors as compared to stimulation with either A P M 2 or C X C L 8 (IL-8). Treatment with reverse peptide AP4 2_i alone (5 uM) had no effect on expression of pro- inflammatory factors compared to control. Furthermore, AP4 2 . i did not alter C X C L 8 (IL- 8) induced expression of pro-inflammatory mediators when AP4 2_i was co-applied with C X C L 8 (IL-8). Effects of A p M 2 , C X C L 8 (IL-8) and of A p M 2 and C X C L 8 (IL-8) combined on anti-inflammatory cytokine expression were also examined (Figure 5-2B). The anti- inflammatory cytokines IL-10 and TGFPi were constitutively expressed under unstimulated conditions and were unaffected by APi_4 2 or C X C L 8 (IL-8) alone, AP1-42 and C X C L 8 (IL-8) combined, AP42-1 alone or AP42-1 in combination with C X C L 8 (IL-8). G3PDH served as a reaction standard (Figure 5-2C). Densitometry analysis of PCR product band intensities indicate a similar effect of C X C L 8 (IL-8) to enhance AP1.42 effects to increase pro-inflammatory mediator expression with no effects on anti-inflammatory cytokine expression. Results are summarized in Figure 5-2D. APi_4 2 and C X C L 8 (IL-8) each alone significantly increased relative mRNA levels of the pro-inflammatory mediators as compared to control (p < 0.05). C X C L 8 (IL-8) in the combined presence of AP1.42 significantly increased relative mRNA levels of all pro-inflammatory mediators as compared to AP1.42 stimulated levels (p < 0.05). Fold increases in relative pro-inflammatory mediator mRNA as a result of ApM2> C X C L 8 (IL-8) and combined AP1.42 and C X C L 8 (IL-8) stimulation compared to levels in control is summarized in Table 5.1. Overall, the fold increases in pro- inflammatory mediators induced by APi_4 2 and C X C L 8 (IL-8) each alone compared to control were: IL- lp : 3.4, 2.7; IL-6: 6.9, 12.2; C X L 8 (IL-8): 1.9, 1.3; TNF-a: 3.2, 1.3; COX-2: 3.0, 2.3. The fold increase in pro-inflammatory mediators induced by Api-42 and C X C L 8 (IL-8) combined compared to A p M 2 alone were: IL- lp : 1.7; IL-6: 2.4; C X C L 8 (IL-8): 1.4; TNF-a: 1.8; COX-2: 2.4. Application of A p 4 2 - i had no effect to alter relative mRNA levels of pro-inflammatory mediators and did not alter relative mRNA levels induced with C X C L 8 (IL-8) when applied in combination (Figure 5-2A). Relative mRNA levels of the anti-inflammatory cytokines IL-10 and TGFPi were unchanged from basal levels despite treatment with stimuli (Figure 5-2D). Figure 5-2. Effects of CXCL8 (IL-8) on APi_42-induced pro inflammatory mediator and anti-inflammatory cytokine expression in human microglia Expression of (A) TNF-a, IL-6, IL-lp, CXCL8 (IL-8), COX-2 and (B) anti- inflammatory cytokines were examined in microglia incubated for 8 hrs with Api-42, CXCL8 (IL-8), A P — in the combined presence of CXCL8 (IL-8), or with medium alone. Stimulation of microglia with Ap 4 2 - i (5 uM) alone or in combination with CXCL8 (IL-8) served as control experiments. The results shown are a representative of five independent experiments. (C) The expression of G3PDH served as a reaction standard. (D) Summary of relative mRNA levels of inflammatory mediators induced by A p M 2 , CXCL8 (IL-8) and combined A p | . 4 2 and CXCL8 (IL-8). Results are expressed as mean ± SEM from n=5 independent experiments. One-way ANOVA and Newman-Keuls multiple comparison post-test was performed to evaluate statistical significance (p < 0.05) (* indicates statistically significant from control; ** indicates statistically significant from Ap — stimulated levels). Table 5-1. Fold increases in relative pro-inflammatory mediator mRNA induced by A p M 2 , CXCL8 (IL-8), A p ^ + CXCL8 (IL-8) compared to relative mRNA in control A P , . 4 2 IL-8 APL42+ IL-8 IL-lp 3.4* 2.7* 5.6*** IL-6 6.9** 12.2*** 16.8*** IL-8 1.9* 1.3* TNF-a 3.2* 1.3* COX-2 3.0* 2.3* 6.9*** * p< 0.05; **p<0.01; ***p<0.001 5.2.3 Effects of CXCL8 (IL-8) on APi^-Induced Production of Pro-inflammatory Mediators I further investigated whether pro-inflammatory factors with increased expression were also increased at the protein level. The production of TNF-a, IL-6, IL-ipand C X C L 8 (IL-8) were investigated after 24 hrs stimulation with AP1-42 in the presence and absence of C X C L 8 (IL-8) using ELISA. The time point 24 hrs was chosen since preliminary findings indicated APi^2 induced optimal production of cytokines at this time point. Incubations with A P M 2 for periods longer than 24hrs could induce both direct and indirect effects of the peptide in human microglia (Walker et al., 2001). A summary of results is presented in Figure 5-3. A B control A P ^ 2 A p ^ 2 IL-8 AP42-1 Ap 42.! + IL-8 + IL-8 c. 200 Figure 5-3. Effects of CXCL8 (IL-8), Api.42 each alone or in combination on pro- inflammatory mediator production Effects of Api.42, CXCL8 (IL-8) and combined A p ^ 2 and CXCL8 (IL-8) on pro- inflammatory cytokine secretion by human microglia using ELISA. Data are mean ± SEM of four independent experiments for (A) TNF-a and three independent experiments for (B) IL-6 (C) IL-ip and (D) CXCL8 (IL-8) each performed in duplicate. Human microglia were exposed to either medium alone, APi_42 (5 uM), CXCL8 (IL-8) (100 ng/mL), A p M 2 in combination with CXCL8 (IL-8), Ap 4 2-i or to Ap 4 2-i in combination with CXCL8 (IL-8) for 24 hrs. One-way ANOVA and Newman-Keuls multiple comparison post-test was performed to evaluate statistical significance (p < 0.05) (* indicates statistically significant from control; ** indicates statistically significant from Api_42 stimulated levels). Low levels of pro-inflammatory cytokines (TNF-a, IL- ip , IL-6) near the detection limits of the assay were produced by human microglia in serum-free medium under basal conditions. The higher level of IL-6 production as compared to TNF-a and IL- ip by microglia under basal conditions was attributed to the use of low serum (1%) in the IL-6 assay whereas serum-free medium was used in TNF-a and IL- ip assays. Fold increases in pro-inflammatory mediator production with APi_42, C X C L 8 (IL-8) and of combined APi_42 and C X C L 8 (IL-8) stimulation compared to control are summarized in Table 5-2. Overall, APi_42 (5 uM) alone significantly increased secretion of TNF-a (by 328%) (Figure 5-3A), IL-6 (by 191%) (Figure 5-3B), IL- lp (by 250%) (Figure 5-3C) and C X C L 8 (IL-8) (by 60%) (Figure 5-3D) all values p < 0.01. C X C L 8 (IL-8) in the combined presence of APi_4 2 significantly enhanced additively the levels of TNF-a (by 79%), IL-6 (by 43%) and IL- lp (by 66%) as compared to A p M 2 stimulated levels (p < 0.05). C X C L 8 (IL-8) (100 ng/mL) alone also increased IL- lp , TNF-a, and IL-6 as compared to control, however, the increases were not significant (p > 0.05). AP42-1 (5 uM) had no effect to increase secreted levels of IL- ip , TNF-a, IL-6 or C X C L 8 (IL-8) compared to unstimulated conditions (p > 0.05). Addition of AP42-1 in combination with C X C L 8 (IL-8) did not significantly increase levels of IL- lp , IL-6 and TNF-a as compared to levels induced with C X C L 8 (IL-8) alone (p > 0.05). Pro-inflammatory cytokine production with Api.42 and C X C L 8 (IL-8) co-stimulation was additive since the sum of pro-inflammatory cytokine production induced with AP1-42 and C X C L 8 (IL-8) each alone was equivalent to the effects of A P i ^ 2 and C X C L 8 (IL-8) applied in combination. Table 5-2. Fold increases in pro-inflammatory mediator production induced by ApY 42, CXCL8 (IL-8), A p — + CXCL8 (IL-8) compared to levels in control A P — IL-8 Api.42+IL-8 IL-lp 3.5* 2.0 5.8** IL-6 2.9* L6 4.2** IL-8 1.7* TNF-a 4.3* 1.3 7 7 * * *p< 0.01; **p<0.0 01 The production of COX-2 after stimulation with A P — , C X C L 8 (IL-8) or A P — and C X C L 8 (IL-8) combined was determined using immunocytochemistry (Figure 5-4). Over the number of COX-2 positive microglia was determined from four representative fields in three independent experiments. A P — significantly increased the percentage of microglia expressing COX-2 by 230% from control levels (p < 0.001) (Figure 5-4B). C X C L 8 (IL-8) increased COX-2 production (Figure 5-4A) compared to unstimulated cells but the increases were not significant (p > 0.05). Co-addition of C X C L 8 (IL-8) in the presence of A p — enhanced additively the percentage of COX-2 positive microglia (by 71%) as compared to A p — levels and the effect was significant (p < 0.001). AP42-1 also increased the percentage of COX-2 positive microglia compared to unstimulated levels but this effect was not significant (p > 0.05). Reverse peptide, AP42-1 applied in combination with C X C L 8 (IL-8) increased COX-2 production as compared to levels induced with C X C L 8 (IL-8) alone but the increase was not significant (p > 0.05). A control A|^42 A|S 1 4 2+ IL-8 COX-2 (green) 4 + 9 * V • ^ v DAPI (blue) m • IL-8 A|l 4 2 . . , , AP42./+IL-8 | • 11 IL-8 Ap42-1 AP42-1 + IL-8 Figure 5-4. Effects of A p — , CXCL8 (IL-8) each alone or in combination on COX-2 expression in human microglia (A) Representative figures of COX-2 positively stained microglia (green) and nuclei of cells (blue). (B) The percentage of COX-2 positive microglia were measured in microglia after 24 hr incubation with 5 uM AP—, 100 ng/mL CXCL8 (IL-8) alone or in combination with Ap—, A p 4 2 i alone or in combination with CXCL8 (IL-8) or with medium alone and data presented as mean ± SEM. Significance was determined using one-way ANOVA and Newman-Keuls multiple comparison post- test (p < 0.05) (* indicates statistically significant from control; indicates statistically significant from Ap — stimulated levels). Scale bar = 50 urn. B control A01.42 APi^ 2 + IL-8 5.3 CONCLUSION The principle novel finding of this aspect of my study is that C X C L 8 (IL-8) enhances Api-42-induced human microglial expression and secretion of pro-inflammatory mediators. Stimulation with A(5i_42 and C X C L 8 (IL-8) together led to significantly enhanced expression (Figure 5-2A) and additive effects to increase production (Figure 5- 3, 5-4) of IL-6, IL- lp , TNF-a and COX-2 as compared to levels with AP1-42 applied alone. Importantly, expression of the anti-inflammatory cytokines EL-10 and TGFPi were unchanged by microglial treatment with peptide or peptide plus C X C L 8 (IL-8) (Figure 5-2B). Overall, the results suggest that C X C L 8 (IL-8) chemokine activity could enhances additively APi^-induced pro-inflammatory responses mediated by activated microglia in A D brain (see below). Overall, the results from semi-quantitative RT-PCR analysis for expression (Figure 5-2) and ELISA analysis for production (Figure 5-3) showed reasonable agreement for C X C L 8 (IL-8) enhancement of AP1.42 stimulation of the inflammatory mediators. However, the results in Fig. 5-2A also show C X C L 8 (IL-8) alone could induce expression of IL-6, TNF-a, IL- lp , C X C L 8 (IL-8) and COX-2. Interestingly, the changes in expression of the pro-inflammatory cytokines and COX-2 by C X C L 8 (IL-8) alone were not accompanied by increased production of the agents (Figure 5-3A-C; 5-4). On the basis of preliminary findings, optimal expression of the pro-inflammatory cytokines were evident with 8 hrs stimulation of microglia, however, at this time point little or no production of cytokines were observed. For production of cytokines, microglia were stimulated for 24 hrs in accord with previous studies using human microglia (Lee et al., 1993). Differences between C X C L 8 (IL-8) effects on expression and production could also reflect tight control of post-transcriptional events in microglia since previous work has shown similar behaviour for this chemokine applied to human neutrophils (Martinez et al., 2004). A previous study has reported that amyloid peptide elicited functional responses in monocytes via a distinct signaling pathway from that induced by chemokines (Badolato et al., 1995). Our results are consistent with separate pathways for C X C L 8 (IL-8) and APi_42-induced stimulation of human microglia. As shown in Figure 5-3A-C (for cytokines) and Figure 5-4 (for COX-2), C X C L 8 (IL-8) applied in combination with AP1.42 produced additive responses. A n interesting possibility is that an autocrine feedback system may play a role in C X C L 8 (IL-8) potentiation of A p ^ 2 effects. In this case, APi_42 mediated release of C X C L 8 (IL-8) (Fig. 5-3D) could stimulate microglia since receptors for this chemokine have been reported in these cells (Lee et al., 2002). As evident in Figure 5-1A, human microglia exposed to serum-free medium exhibit a general profile of ramified morphology. After exposure of human microglia to Api_425 cells show a trend to ameboid morphology (Figure 5-1C). This result is consistent with results from previous work where treatment of microglia with A P M 2 caused a shift from ramified to ameboid shape (Walker et al., 2001). Interestingly, in the present study I observed no changes in cell morphology after exposure to C X C L 8 (IL-8) treatment where cells exhibited a predominant ramified shape similar to unstimulated conditions (Figure 5-IB). Since C X C L 8 (IL-8) induced the expression of cytokines, our results suggest some dissociation between morphology and priming effects of C X C L 8 (IL-8). This point is important since ramified and ameboid morphologies have been considered as indicative of resting and activated states of microglia (Ling and Wong, 1993). Previous work has demonstrated APi^-induced microglial expression of pro- inflammatory cytokines (Lue et al., 2001a; Walker et al., 1995; 2001) and COX-2 (Hoozemans et al., 2001; 2002b). Reports by Lee et al., 2002 and Nagai et al., 2001 indicate that unstimulated human microglia in a high serum (5 % horse serum) containing medium express basal levels of IL- ip , IL-6 and TNF-a and a prominent ameboid morphology. Since I found no basal expression of pro-inflammatory cytokines and a ramified morphology in control, it seems likely that differences could reflect our use of serum-free medium. The lack of effect of stimuli including amyloid peptide on expression of the anti-inflammatory cytokines IL-10 and TGFPi in microglia (Figure 5- 2B) in the present study are in agreement with previous reports (Meda et al., 1999; Walker et al., 2001). These results would indicate that although microglia exhibit a pro- inflammatory profile as a result of amyloid peptide stimulation in the presence or absence of C X C L 8 (IL-8), their anti-inflammatory properties are preserved. Enhancement of Ap responses has also been reported previously with the agents interferon-y (Meda et al., 1996), macrophage colony stimulating factor (M-CSF) (Murphy et al., 1998), C l q and serum amyloid P (Veerhuis et al., 2003). However, available evidence indicates that microglia produce little or no amounts of these factors (McGeer and McGeer, 1995; Yasojima et al., 2000; Lue et al., 2001a). Our results have particular significance to inflammatory responses mediated by microglia in A D . C X C L 8 (IL-8) has been observed as the most prominent factor expressed by adult human microglia stimulated with AP1-42 (Walker et al., 2001) and C X C L 8 (IL-8) is highly elevated in A D brain (Galimberti et al., 2003). Taken together, these findings suggest that autocrine release of C X C L 8 (IL-8) from microglia enhances Api_42 stimulation of cells and that this cytokine could function to exacerbate inflammatory responses in A D brain. The inhibition of C X C L 8 (IL-8) actions could constitute an effective strategy for therapeutic intervention in slowing the progression of A D . Chapter 6: DISCUSSION OF THESIS RESEARCH AND FUTURE DIRECTIONS The studies presented in this thesis focused on determining the effects of acute and chronic treatment with full length, AP1.42, on intracellular signaling pathways and functional responses of microglia using electrophysiology, calcium spectrofluorometry, RT-PCR, ELISA, immunocytochemistry and immunohistochemistry techniques. The hypothesis of my research was that inhibition of APi^-induced microglial mediated intracellular signaling pathways would downregulate inflammatory functional responses of microglia which could serve as a therapeutic strategy for A D . The results presented in this thesis support this hypothesis. I first investigated the effects of AP1.42 on human microglial membrane potential and membrane current expression using electrophysiology. APi_42 acutely applied to human microglia induced the expression of a novel outward K + current which' was sensitive to the non-selective K + channel blocker 4-aminopyridine (4-AP). A current similar in properties was observed with intracellular application of the non-hydrolyzable analogue of GTP, GTPyS. This would suggest that the outward K + current induced by acute APi.42 was induced via a G protein. Molecular biology studies indicated that the K + channel induced by APi_42 was likely due to Kv3.1. Also, A P M 2 increased the expression of the Fcyll receptor. Other studies using electrophysiology indicated that acute application of AP1-42 induced a transient depolarization in human microglia; inhibition of the Fcyll receptor inhibited this depolarization suggesting a link between the Fcyll receptor and APi_4 2 effects on cell potential. From these studies, I established that AP1.42 alters membrane current expressions in microglia and that 4-AP could serve as a potential modulator of APi_42-induced microglial inflammatory and neurotoxic responses. I then examined the effects of 4-AP on AP1.42 induced Ca responses in human microglia using the calcium spectrofluorometry technique. I was able to show that A p ^ 2 induces an increase in [Ca ]; through an unidentified influx pathway. 4-AP inhibited this C a 2 + influx pathway activated by acute APM2- These result would indicate that the unidentified C a 2 + influx pathway induced by acute APi_42 hi human microglia is sensitive to membrane potential. Using a series of in vitro (RT-PCR, ELISA, immunocytochemistry) and in vivo assays (immunohistochemistry), the effects of APi_42 on functional responses of human microglia including potential neurotoxicity was investigated. In vitro, I was able to show that 4-AP inhibited APi_42 effects to activate p38 M A P kinase, N F K B , the expression and production of pro-inflammatory cytokines IL - ip , IL-6, TNF-a, me chemokine C X C L 8 (IL-8) and inducible enzyme COX-2 as well as microglial mediated neurotoxicity. More importantly, 4-AP reduced neuronal damage and microglial activation induced by A P M 2 in vivo which strongly supports the in vitro actions of 4-AP as a modulator of AP1.42 mediated pro-inflammatory responses. These results suggest that 4-AP modulates AP1-42- induced intracellular signaling pathways and functional responses in human microglia including microglial-mediated neurotoxicity. The final set of studies examined the effects of the chemokine C X C L 8 (IL-8) on APi_42-induced expression and production of the pro-inflammatory cytokines IL-6, IL - lp , TNF-a, the inducible enzyme COX-2 and chemokine C X C L 8 (IL-8). Microglial treatment with C X C L 8 (IL-8) added with ApM2 led to enhancement in both expression and production of all of these pro-inflammatory factors compared with peptide alone. The expression of the anti-inflammatory cytokines IL-10 and TGFPi remained unchanged from basal levels with stimulation using either APi_42, C X C L 8 (EL-8) or the peptide together with C X C L 8 (IL-8). Since C X C L 8 (IL-8) is elevated in A D brain and highly expressed by AP stimulated microglia, these results would suggest that other factors such as C X C L 8 (IL-8) could be acting in concert with AP1-42 to additively enhance inflammatory responses by microglia in A D . The results summarized above illustrate that inflammatory stimuli such as AP1.42 have profound signaling effects on microglia and that modulating these intracellular signaling pathways can either inhibit (as shown with 4-AP) or potentiate (as shown with C X C L 8 (IL-8)) the pro-inflammatory effects of microglia. These results offer further insight into the interactions that microglia have with surrounding stimuli, the importance of particular intracellular signaling pathways in mediating microglial responses and most importantly, how modulation of these signaling pathways inhibits microglial inflammatory responses. The significance of these results is that modulating microglial mediated inflammatory functional responses to stimuli such as A P M 2 with agents that inhibit intracellular signaling pathways could serve as a therapeutic strategy for use in neurodegenerative diseases such as A D . Results from this thesis work indicate that 4-AP would be a suitable candidate for the treatment of A D . 4-AP has favourable properties over other K + channel inhibitors since it is able to cross the B B B . It should be noted that 4-AP has been used clinically for the treatment of A D with some positive results. However, careful consideration must be taken in its use clinically since 4-AP has unwanted side effects most notably at higher doses. Thus, 4-AP could be used at low doses or as an adjunctive therapy in the treatment of A D . Further studies on the potential use of specific K + channel blockers, such as a specific blocker of Kv3.1, in inflammatory diseases of the CNS should also be considered. Future research could also be carried out to determine the nature of the C a 2 + influx pathway induced by A p — in human microglia. It would also be of interest to determine the role of the FcyRII receptor in mediating microglial inflammatory responses and whether inhibition of the FcyRII receptor could serve as potential modulator of microglial functional responses. Since several intracellular factors are implicated in 9-+- linking A p — to functional responses of microglia including [Ca p38 M A P K and N F K B , each of these factors could serve as potential modulatory sites of microglial functional responses. Furthermore, an inhibitor of C X C L 8 (IL-8) could be used as a modulator of microglial mediated inflammatory responses in A D . Moreover, the potential modulatory actions of combination therapies i.e. a K + channel inhibitor in the presence of another modulator of Ap—-induced intracellular signaling, could be investigated using in vitro assays of Ap—-induced microglial mediated inflammatory responses as well as in transgenic A D animals. The positive results reported in this thesis of modulating microglial mediated inflammatory responses both in vitro and in vivo by using an inhibitor of stimulus- induced signaling will hopefully lead to further studies on potential modulators of intracellular signaling pathways in microglia. Ultimately, it is hoped that potential modulators of microglial mediated inflammatory responses with significant beneficial effects in vitro and in vivo will be developed for application in a clinical setting. R E F E R E N C E S Agdeppa, E. D., Kepe, V . , Petric, A. , Satyamurthy, N . , Liu, J., Huang, S.-C, Small, G. W., Cole, G. M . , Barrio, J. R. 2003. In vitro detection of (S)-naproxen and ibuprofen binding to plaques in the Alzheimer's brain using the positron emission tomography molecular imaging probe 2-(l-6[(2-[18F]fluoroethyl)(memyl)amino]-2- nnaphtylethylidene)malononitrile. Neurosci. 117: 723-730. Aisen, P.S. 2002. The potential of anti-inflammatory drugs for the treatment of Alzheimer's disease. Lancet Neurol. 1:279-284. Aisen, P.S., Davis, K . L . , Berg, J.D., Schafer, K. , Campbell, K. , Thomas, R.G., Weiner, M.F., Farlow, M.R., Sano, M . , Grundman, M . , Thai, L.J. 2000. A randomized controlled trial of prednisone in Alzheimer's disease: Alzheimer's Disease Cooperative Study. Neurol. 54:588-593. Akiyama, H. , Barger, S., Barnum, S., Bradt, B. , Bauer, J., Cole, G .M. , Cooper, N.R., Eikelenboom, P., Emmerling, M . , Fiebich, B.L. , Finch, C.E., Frautschy, S., Griffin, W.S., Hampel, H. , Hull, M . , Landreth, G., Lue, L. , Mrak, R , MacKenzie, I.R., McGeer, P.L., O'Banion, M . K . , Pachter, J., Pasinetti, G., Plata-Salaman, C , Rogers, J., Rydel, R , Shen, Y . , Streit, W., Strohmeyer, R., Tooyoma, I., Van Muiswinkel, F.L., Veerhuis, R., Walker, D., Webster, S., Wegrzyniak, B., Wenk, G., Wyss-Coray, T. 2000. Inflammation and Alzheimer's disease. Neurobiol. Aging 21: 383-421. Akiyama, H. , McGeer, P.L. 1990. Brain microglia constitutively express beta-2 integrins. J Neuroimmunol. 30:81-93. Allan, S.M., Rothwell, N.J . 2001. Cytokines and acute neurodegeneration. Nat. Rev. Neurosci. 2:734-744. Ambrosini, E. and Aloisi, F. 2004. Chemokines and glial cells: a complex network in the central nervous system. Neurochem. Res. 29:1017-1038. Anandatheerthavarada, H.K., Biswas, G., Robin, M . A . , Avadhani, N .G . 2003. Mitochondrial targeting and a novel transmembrane arrest of Alzheimer's amyloid precursor protein impairs mitochondrial function in neuronal cells. J. Cell. Biol. 161:41- 54. Armstrong, R.A. 1998. Beta-amyloid plaques: stages in life history or independent origin? Dement. Geriatr. Cogn. Disord. 9:227-238. Arnold, S.E., Hyman, B.T., Flory, J., Damasio, A.R., Van, Hoesen, G.W. 1991. The topographical and neuroanatomical distribution of neurofibrillary tangles and neuritic plaques in the cerebral cortex of patients with Alzheimer's disease. Cereb. Cortex. 1:103- 116. Arriagada , P.V, Marzloff, K. , Hyman, B.T. 1992. Distribution of Alzheimer-type pathologic changes in nondemented elderly individuals matches the pattern in Alzheimer's disease. Neurol. 42:1681-1688. Atwood, C.S., Moir, R.D., Huang, X . , Scarpa, R.C., Bacarra, N . M . , Romano, D . M . , Hartshorn, M.A . , Tanzi, R.E., Bush, A.I. 1998. Dramatic aggregation of Alzheimer abeta by Cu(II) is induced by conditions representing physiological acidosis. J. Biol . Chem. 273:12817-12826. Badolato, R., Johnston, J.A., Wang, J .M. , McVicar, D., Xu , L .L . , Oppenheim, J.J., Kelvin, D.J. 1995. Serum amyloid A induces calcium mobilization and chemotaxis of human monocytes by activating a pertussis toxin-sensitive signaling pathway. J. Immunol. 155:4004-4010. Bamberger, M.E. , Harris, M.E. , McDonald, D.R., Husemann, J., Landreth, G.E. A cell surface receptor complex for fibrillar beta-amyloid mediates microglial activation. J. Neurosci. 23:2665-2674. Bamberger, M.E. , Landreth, G.E. 2001. Microglial interaction with beta-amyloid: implications for the pathogenesis of Alzheimer's disease. Microsc. Res. Tech. 54:59-70. Banati, R.B. and Graeber, M . B . 1994. Surveillance, intervention and cytotoxicity: is there a protective role of microglia? Dev. Neurosci.16:114-127. Bard, F., Cannon, C , Barbour, R., Burke, R.L., Games, D., Grajeda, H. , Guido, T., Hu, K. , Huang, J., Johnson-Wood, K. , Khan, K. , Kholodenko, D., Lee, M . , Lieberburg, I., Motter, R., Nguyen, M . , Soriano, F., Vasquez, N . , Weiss, K. , Welch, B., Seubert, P., Schenk, D., Yednock, T. 2000. Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease. Nat Med. 6:916-919. Barger, S.W. and Basile, A.S. 2001. Activation of microglia by secreted amyloid precursor protein evokes release of glutamate by cystine exchange and attenuates synaptic function. J Neurochem. 76:846-854. Barone, F.C., Arvin, B. , White, R.F., Miller, A . , Webb, C.L., Willette, R.N. , Lysko, P.G., Feuerstein, G.Z. 1997. Tumor necrosis factor-alpha. A mediator of focal ischemic brain injury. Stroke. 28:1233-1244. Beher, D., Hesse, L. , Masters, C. L. , Multhaup, G. 1996. Regulation of amyloid protein precursor (APP) binding to collagen and mapping of the binding sites on APP and collagen type I. J. Biol. Chem. 271:1613-1620. Benveniste, E .N. 1992. Inflammatory cytokines within the central nervous system: sources, function and mechanisms of action. Am. J. Physiol. 263:C1-C6. Benveniste, E.N. , Nguyen, V.T., O'Keefe, G . M . 2001. Immunological aspects of microglia: relevance to Alzheimer's disease. Neurochem Int. 2001 39:381-391. Bitting, L. , Naidu, A. , Cordell, B. , Murphy, G . M . Jr. 1996. Beta-amyloid peptide secretion by a microglial cell line is induced by beta-amyloid-(25-35) and lipopolysaccharide. J. Biol. Chem. 271:16084-16089. Blasko, I., Apochal, A . , Boeck, G., Hartmann, T., Grubeck-Loebenstein, B. Ransmayr, G. 2001. Ibuprofen decreases cytokine-induced amyloid beta production in neuronal cells. Neurobiol. Dis. 8, 1094-1101. Blasko, I., Marx, F., Steiner, E., Hartmann, T., Grubeck-Loebenstein, B. 1999. TNFalpha plus IFNgamma induce the production of Alzheimer beta-amyloid peptides and decrease the secretion of APPs. FASEB J. 13:63-68. Boehme, S.A., Lio, F .M. , Maciejewski-Lenoir, D., Bacon, K .B . , Conlon, P.J. 2000. The chemokine fractalkine inhibits Fas-mediated cell death of brain microglia. J. Immunol. 165:397-403. Boireau, A. , Richard, F., Olivier, V . , Aubeneau, M . , Miquet, J .-M., Dubedat, P., Laduron, P., Doble, A . and Blanchard, J.-C. 1991. Differential effects of potassium channel blockers on dopamine release from striatal slices. J. Pharm. Pharmacol. 43: 798-801. Boland, K. , Behrens, M . , Choi, D., Manias, K. , Perlmutter, D.H. 1996. The serpin- enzyme complex receptor recognizes soluble, nontoxic amyloid-beta peptide but not aggregated, cytotoxic amyloid-beta peptide. J. Biol . Chem. 271:18032-18044. Bonaiuto, C , McDonald, P.P., Rossi, F., Cassatella, M.A.I997. Activation of nuclear factor-kappa B by beta-amyloid peptides and interferon-gamma in murine microglia. J Neuroimmunol. 77:51-56. Bordey, A. , Sontheimer, H. 1999. Differential inhibition of glial K(+) currents by 4-AP. J Neurophysiol. 82:3476-87. Brenman, J.E., Bredt, D.S. 1997. Synaptic signaling by nitric oxide. Curr. Opin. Neurobiol. 7:374-378. Bronner, C , Cothenet, V . , Monte, D., Joseph, M . , Landry, Y. , Capron, A . 1990. Role of phospholipase A2 and G-proteins in the IgE-dependent activation of mast cells and macrophages. Agents Actions. 30:95-97. Bruce-Keller, A.J . , Keeling, J.L., Keller, J.N., Huang, F.F., Camondola, S., Mattson, M.P. 2000. Antiinflammatory effects of estrogen on microglial activation. Endocrinol. 141:3646-3656. Buxbaum, J.D., Oishi, M . , Chen, H.I., Pinkas-Kramarski, R., Jaffe, E.A., Gandy, S.E., Greengard, P. 1992. Cholinergic agonists and interleukin 1 regulate processing and secretion of the Alzheimer beta/A4 amyloid precursor protein. PNAS 89:100075- 100078. Buxbaum, J. D., Geoghagen, N . S., Friedhoff, L . T. 2001. Cholesterol depletion with physiological concentrations of a statin decreases the formation of the Alzheimer amyloid Abeta peptide. J. Alzheimers Dis. 3:221-229. Caggiano, A.O. and Kraig, R.P. 1998. Prostaglandin E2 and 4-aminopyridine prevent the lipopolysaccharide-induced outwardly rectifying potassium current and interleukin-ip production in cultured rat microglia. J. Neurochem. 70:2357-2368. Caporaso, G.L., Takei, K. , Gandy, S.E., Matteoli, M . , Mundigl, O., Greengard, P., De Camilli, P. 1994. Morphologic and biochemical analysis of the intracellular trafficking of the Alzheimer beta/A4 amyloid precursor protein. J. Neurosci. 14:3122-3138. Casamenti, F., Corradetti, R., Loffelholz, K. , Mantovani, P., Pepeu, G. 1982. Effects of 4- aminopyridine on acetylcholine output from the cerebral cortex of the rat in vivo. Br J Pharmacol. 76:439-445. Caserta, M.T., Caccioppo, D., Lapin, G.D., Ragin, A. , Groothuis, D.R. 1998. Blood-brain barrier integrity in Alzheimer's disease patients and elderly control subjects. J. Neuropsychiatry Clin. Neurosci. 10:78-84. Chandy, K .G . , Cahalan, M.D. , Grissmer, S. 1990. Autoimmune diseases linked to abnormal K + channel expression in double-negative CD4"CD8" T cells. Eur. J. Immunol. 20:747-751. Chao, C.C., Hu, S. 1994. Tumor necrosis factor-alpha potentiates glutamate neurotoxicity in human fetal brain cell cultures. Dev. Neurosci. 16:172-179. Chao, C.C., Hu, S., Sheng, W.S., Peterson, P.K. 1995. Tumor necrosis factor-alpha production by human fetal microglial cells: regulation by other cytokines. Dev. Neurosci. 17:97-105. Chao, C.C., Molitor, T.W., Hu, S. 1993. Neuroprotective role of IL-4 against activated microglia. J. Immunol. 151:1473-1481. Chapman, P.F., Falinska, A . M . , Knevett, S.G., Ramsay, M.F. 2001. Genes, models and Alzheimer's disease. Trends Genet 17:254-261. Chartier-Harlin, M.C. , Crawford, F., Houlden, H. , Warren, A . , Hughes, D., Fidani, L. , Goate, A . , Rossor, M . , Roques, P., Hardy, J. 1991. Early-onset Alzheimer's disease caused by mutations at codon717 of the beta-amyloid precursor protein gene. Nature 353:844-846. Chattopadhyay, N . , Ye, C , Yamaguchi, T., Nakai, M . , Kifor, O., Vassilev, P .M. , Nishimura, R.N. , Brown, E . M . 1999. The extracellular calcium-sensing receptor is expressed in rat microglia and modulates an outward K+ channel. J Neurochem. 72:1915- 22. Cheng, B. , Christakos, S., Mattson, M P . 1994. Tumor necrosis factors protect neurons against metabolic-excitotoxic insults and promote maintenance of calcium homeostasis. Neuron. 12:139-153. Choi, H.B., Hong, S.H., Ryu, J.K., Kim, S.U., McLarnon, J.G. 2003. Differential activation of subtype purinergic receptors modulates C a 2 + mobilization and COX-2 in human microglia. Glia. 43, 95-103. Christman, J.W., Blackwell, T.S., Juurlink, B .H. 2000. Redox regulation of nuclear factor kfi: therapeutic potential for attenuating inflammatory responses. Brain Path. 10: 153-162. Chung, S., Joe, E., Soh, H. , Lee, M . Y . , Bang, H.W. 1998. Delayed rectifier potassium currents induced in activated rat microglia set the resting membrane potential. Neurosci Lett. 242:73-76. Chung, S., Lee, J., Joe, E.H., Uhm, D.Y. 2001. Beta-amyloid peptide induces the expression of voltage dependent outward rectifying K + channels in rat microglia. Neurosci. Lett. 300:67-70. Citron, M . , Oltersdorf, T., Haass, C , McConlogue, L. , Hung, A . Y . , Seubert, P., Vigo- Pelfrey, C , Lieberburg, I., Selkoe, D.J. 1992. Mutation of the beta-amyloid precursor protein in familial Alzheimer's disease increases beta-protein production. Nature 360:672-674. Citron, M . , Westaway, D., Xia , W., Carlson, G., Diehl, T., Levesque, G., Johnson-Wood, K. , Lee, M . , Seubert, P., Davis, A . , Kholodenko, D., Motter, R., Sherrington, R., Perry, B., Yao, H. , Strome, R., Lieberburg, I., Rommens, J., Kim, S., Schenk, D., Fraser, P., St. George Hyslop, P., Selkoe, D.J. 1997. Mutant presenilins of Alzheimer's disease increase production of 42-residue amyloid beta-protein in both transfected cells and transgenic mice. Nat. Med. 3:67-72. Claudio, L . 1996. Ultrastructural features of the blood-brain barrier in biopsy tissue from Alzheimer's disease patients. Acta Neuropathol. 91:6-14. Colton, C.A., Chernyshev, O.N., Gilbert, D.L., Vitek, M P . 2000. Microglia contribution to oxidative stress in Alzheimer's disease. Ann. N . Y . Acad. Sci. 899:292-307. Colton, C A . and Gilbert, D.I. 1987. Production of superoxide anions by a CNS macrophage, the microglia. FEBS Lett. 223:284-288. Combs, C.K., Johnson, D.E., Cannady, S.B., Lehman, T .M. , Landreth, G.E. 1999. Identification of microglial signal transduction pathways mediating a neurotoxic response to amyloidogenic fragments of beta-amyloid and prion proteins. J. Neurosci. 19:928-939. Combs, C.K., Johnson, D.E., Karlo, J.C., Cannady, S.B., Landreth, G.E. 2000. Inflammatory mechanisms in Alzheimer's disease: inhibition of beta-amyloid-stimulated proinflammatory responses and neurotoxicity by PPARgamma agonists. J Neurosci. 20:558-567. Combs, C.K., Karlo, J.C., Kao, S.C., Landreth, G.E. 2001. beta-Amyloid stimulation of microglia and monocytes results in TNFalpha-dependent expression of inducible nitric oxide synthase and neuronal apoptosis. J. Neurosci. 21:1179-1188. Cooper, N.R., Bradt, B . M . , O'Barr, S., Yu , J.X. 2000. Focal inflammation in the brain: role in Alzheimer's disease. Immunol Res. 21:159-165. Corder, E.H., Saunders, A . M . , Strittmatter, W.J., Schmechel, D.E., Gaskell, P.C., Small, G.W., Roses, A.D. , Haines, J.L., Pericak-Vance, M . A . 1993. Gene dose of apolipoprotein E type £4 allele and the risk of Alzheimer's disease in late onset families. Science. 261: 921-923. Costa, M . M . , Reus, V.I., Wolkowitz, O.M., Manfredi, F., Lieberman, M . 1999. Estrogen replacement therapy and cognitive decline in memory-impaired post-menopausal women. Biol. Psychiatry 46:182-188. Cotman, C.W., and Anderson, A.J . 1995. A potential role for apoptosis in neurodegeneration and Alzheimer's disease. Mol . Neurobiol. 10:19-45. Cotman, C.W., Tenner, A.J . , Cummings, B.J. 1996. B-amyloid converts an acute phase injury response to chronic injury responses. Neurobiol. Aging 17:723-731. Cross, A . K . , Woodroofe, M . N . 1999. Chemokines induce migration and changes in actin polymerization in adult rat brain microglia and a human fetal microglial cell line in vitro. J. Neurosci. Res. 55:17-23. Cummings, B.J., Pike, C.J., Shankle, R., Cotman, C.W. 1996. Beta-amyloid deposition and other measures of neuropathology predict cognitive status in Alzheimer's disease. Neurobiol. Aging 17: 921-933. Cummings, J.L., Vinters, H.V. , Cole, G .M. , Khachaturian, Z.S. 1998. Alzheimer's disease. Neurology. 51 :S2-S 17. D'Andrea, M.R., Nagele, R.G., Wang, H.Y. , Peterson, P.A., Lee, D.H. 2001. Evidence that neurones accumulating amyloid can undergo lysis to form amyloid plaques in Alzheimer's disease. Histopathology. 38:120-134. Davidson, M . , Zemishlany, Z., Mohs, R.C., Horvath, T.B., Powchik, P., Blass, J.P., Davis, K . L . 1988. 4-Aminopyridine in the treatment of Alzheimer's disease. Biol Psychiatry. 23:485-90. Davis, J.B., McMurray, H.F., Schubert, D. 1992. The amyloid beta-protein of Alzheimer's disease is chemotactic for mononuclear phagocytes. Biochem. Biophys. Res. Commun. 189:1096-1100. Dawson, G.R., Seabrook, G.R., Zheng, H. , Smith, D.W., Graham, S., O'Dowd, G., Bowery, B.J., Boyce, S., Trumbauer, M.E. , Chen, H.Y. , Van der Ploeg, L.H.T., Sirinathsingji, J.S. 1999. Age-related cognitive deficits, impaired long-term potentiation and reduction in synaptic marker density in mice lacking the beta-amyloid precursor protein. Neurosci. 90:1-13. de la Monte, S.M., Luong, T., Neely, T.R., Robinson, D., Wands, J.R. 2000. Mitochondrial D N A damage as a mechanism of cell loss in Alzheimer's disease. Lab Invest. 80:1323-1335. del Rio-Hortega, P. 1932 Microglia. In: Cytology and cellular pathology of the nervous system. New York: P.B. Hoeber,- 483-534. Deutseh, C. 2002. Potassium channel ontogeny. Ann. Rev. Physiol. 64:19-46. Dickson, D.W. 2004. Apoptotic mechanisms in Alzheimer neurofibrillary degeneration: cause or effect? J. Clin. Invest. 114:23-27. Dickson, D.W., Lee, S.C., Mattiace, L .A. , Yen, S.-H.C, Brosnan C.F. 1993. Microglia and cytokines in neurological disease, with special reference to AIDS and Alzheimer's disease. Glia 7:75-83. Dickson, T.C., Vickers, J.C. 2001. The morphological phenotype of beta-amyloid plaques and associated neuritic changes in Alzheimer's disease. Neuroscience 105:99-107. Dinarello C A . 1997a. Blocking interleukin-1 and tumor necrosis factor in disease. Eur Cytokine Netw. 8: 294-296. Dinarello CA. 1997b. Induction of interleukin-1 and interleukin-1 receptor antagonist. Semin. Oncol. 24: S9-S9. Dodel, R.C., Du, Y . , Bales, K.R., Gao F., Paul, S.M. 1999. Sodium salicylate and 17(3- estradiol attenuate nuclear transcription factor NF-kappaB translocation in cultured rat astroglial cultures following exposure to amyloid Ap (1-40) and lipopolysaccharides. J. Neurochem. 73:1453-1460. Dolezal, V . and Tucek, S. 1983. The effects of 4-aminopyridine and tetrodotoxin on the release of acetylcholine from rat striatal slices. Naunyn-Schmiedeberg's Arch. Pharmacol. 323: 90-95. Dopp, J .M., Mackenzie-Graham, A. , Otero, G.C., Merrill, J.E. 1997. Differential expression, cytokine modulation, and specific functions of type-1 and type-2 tumor necrosis factor receptors in rat glia. J. Neuroimmunol. 75:104-112. Dovey, H. , Varghese, J., Anderson, J.P. 2000. Functional gamma-secretase inhibitors reduce beta-amyloid peptide levels in the brain. J. Neurochem. 76:1-10. DuBourdieu, D.J., Morgan, D.W. 1990. Multiple pathways for signal transduction in the regulation of arachidonic acid metabolism in rat peritoneal macrophages. Biochim. Biophys. Acta. 1054:326-332. Edbauer, D., Winkler, E., Regula, J.T., Pesold, B. , Steiner, H. , Haass, C. 2003. Reconstitution of gamma-secretase activity. Nat. Cell. Biol. 5:486-488. Eder, C. 1998. Ion channels in microglia (brain macrophages): Am. J. Physiol. 275:C327-342. Egger, T., Schuligoi, R., Wintersperger, A . , Amann, R., Malle, E., Sattler, W. 2003. Vitamin E (alpha-tocopherol) attenuates cyclo-oxygenase 2 transcription and synthesis in immortalized murine BV-2 microglia. Biochem J. 370:459-67. Ehrlich, L.C. , Hu, S., Sheng, W.S., Sutton, R.L., Rockswold, G.L., Peterson, P.K., Chao, C.C. 1998. Cytokine regulation of human microglial cell IL-8 production. J. Immunol. 160, 1944-1948. Eikelenboom, P., Rozemuller, J .M., van Muiswinkel, F X . 1998. Inflammation and Alzheimer's disease: relationships between pathogenic mechanisms and clinical expression. Exp. Neurol. 154:89-98. E l Khoury, J., Hickman, S.E., Thomas, C.A., Cao, L. , Silverstein, S.C., Loike, J.D. 1996. Scavenger receptor-mediated adhesion of microglia to beta-amyloid fibrils. Nature. 382:716-719. Ertekin-Taner, N . , Ronald, J., Younkin, L .H . , Hella, M . , Jain, S., Hackett, A . , Scanlin, L. , Kelly, J., Hutton, M . , Younkin, S.G. 2002. Urokinase plasminogen activator haplotypes are significantly associated with Abeta42 and late-onset Alzheimer's disease. Neurobiol. Ag. 23:S314. Felts, P.A. and Smith, K.J . 1994. The use of potassium channel blocking agents in the therapy of demyelinating diseases. Ann. Neurol. 36:454. Fillit, H . , Ding, W.H., Buee, L. , Kalman, J., Altstiel, L . , Lawlor, B. , Wolf-Klein, G. 1991. Elevated circulating TNF levels in Alzheimer's disease. Neurosci Lett 129: 318-320. Fischer, H.G., Eder, C , Hadding, U . , Heineman, U . 1995. Cytokine-dependent K+ channel profile of microglia at immunologically defined functional states. Neurosci. 64:183-191. Flanders, K .C. , Ren, R.F., Lippa, C.F. 1998. Transforming growth factor-betas in neurodegenerative disease. Prog. Neurobiol. 54:71-85. Franciosi, S., Choi, H.B., Kim, S.U., McLarnon, J.G. 2002. Interferon-gamma acutely induces calcium influx in human microglia. J. Neurosci. Res. 69:607-613. Frautschy, S.A., Cole, G .M. , Baird, A . 1992. Phagocytosis and deposition of vascular beta-amyloid in rat brains injected with Alzheimer beta-amyloid. Am. J. Pathol. 140:1389-1399. Frautschy, S.A., Yang, F., Irrizarry, M . , Hyman, B., Saido, T.C., Hsiao, K. , Cole, G . M . 1998. Microglial response to amyloid plaques in APPsw transgenic mice. Am. J. Pathol. 152:307-317. Gabuzda, D., Busciglio, J., Chen, L.B. , Matsudaira, P., Yankner, B.A. 1994. Inhibition of energy metabolism alters the processing of amyloid precursor protein and induces a potentially amyloidogenic derivative. J. Biol. Chem. 269:13623-13628. Gahtan E, Overmier JB. 1999. Inflammatory pathogenesis in Alzheimer's disease: biological mechanisms and cognitive sequeli. Neurosci. Biobehav. Rev. 23:615-633. Galimberti, D., Schoonenboom, N . , Scarpini, E., Scheltens, P. 2003. Chemokines in serum and cerebrospinal fluid of Alzheimer's disease patients. Ann. Neurol. 53, 547-548. Gan, L. , Perney, T .M. , Kaczmarek, L .K . 1996. Cloning and characterization of the promoter for a potassium channel expressed in high frequency firing neurons. J. Biol . Chem. 271: 5859-5865. Gao, Y . , Pimplikar, S.W. 2001. The gamma -secretase-cleaved C-terminal fragment of amyloid precursor protein mediates signaling to the nucleus. PNAS. 98:14979-14984. Gao, J., Wu, D., Guo, T.B., Ruan, Q., L i , T., Lu, Z., Xu , M . , Dai, W., Lu, L . 2004. K + channel activity and redox status are differentially required for JNK activation by U V and reactive oxygen species. Exp. Cell Res. 297:461-471. Garthwaite, J. 1991. Glutamate, nitric oxide and cell-cell signalling in the nervous system. Trends Neurosci. 14:60-67. Gary, D.S., Bruce-Keller, A.J . , Kindy, M.S., Mattson, M.P. 1998. Ischemic and excitotoxic brain injury is enhanced in mice lacking the p55 tumor necrosis factor receptor. J. Cereb. Blood FlowMetab. 18:1283-1287. Gehrmann, J., Matsumoto, Y . , Kreutzberg, G.W. 1995. Microglia: intrinsic immunoeffector cell of the brain. Brain Res. Rev. 20: 269-287. Gibson, G.E., Sheu, K.F. , Blass, J.P. 1998. Abnormalities of mitochondrial enzymes in Alzheimer disease. J. Neural. Transm. 105:855-870. Giovannini, M.G. , Scali, C., Prosperi, C., Bellucci, A . , Vannucchi, M.G . , Rosi, S., Pepeu, G., Casamenti, F. 2002. Beta-amyloid-induced inflammation and cholinergic hypofunction in the rat brain in vivo: involvement of the p38MAPK pathway. Neurobiol. Dis. 11:257-274. Giulian, D. 1997. Immune responses and dementia. Ann. N . Y . Acad. Sci. 835:91-110. Giulian, D., Haverkamp, L.J. , L i , J., Karshin, W.L., Yu , J., Tom, D., L i , X . , Kirkpatrick, J.B. 1995. Senile plaques stimulate microglia to release a neurotoxin found in Alzheimer brain. Neurochem. Int. 27:119-137. Giulian, D., L i , J., Leara, B., Keenen, C. 1994. Phagocytic microglia release cytokines and cytotoxins that regulate the survival of astrocytes and neurons in culture. Neurochem. Int. 25:227-233. Giulian, D., Vaca, K. , Corpuz, M . 1993. Brain glia release factors with opposing actions upon neuronal survival. J. Neurosci. 13:29-37. Glover, W.E. 1982. The aminopyridines. Gen. Pharmacol. 13:259-285. Goate, A. , Chartier-Harlin, M.C. , Mullan, M . , Brown, J., Crawford, F., Fidani, L. , Giuffra, L. , Haynes, A . , Irving, N . , James, L. , 1991. Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease. Nature 349:704-706. Golde, S.? Chandran, S., Brown, G.C., Compston, A . 2002. Different pathways for iNOS- mediated toxicity in vitro dependent on neuronal maturation and N M D A receptor expression. J. Neurochem. 82:269-282. Golde, T.E. 2003. Alzheimer disease therapy: can the amyloid cascade be halted? J. Clin. Invest. 111:11-18. Goldgaber, D., Harris, H.W., Hla, T., Maciag, T., Donnelly, R.J., Jacobsen, J.S., Vitek, M.P., Gajdusek, D.C. Interleukin 1 regulates synthesis of amyloid beta-protein precursor mRNA in human endothelial cells. PNAS. 86:7606-7610. Goodman, Y . , Mattson, M.P. 1994. Secreted forms of beta-amyloid precursor protein protect hippocampal neurons against amyloid beta-peptide-induced oxidative injury. Exp Neurol. 128:1-12. Griffin, W.S.T. and Mrak, R.E. 2002. Interleukin-1 in the genesis and progression of and risk for development of neuronal degeneration in Alzheimer's disease. J. Leuk. Biol. 72, 233-238. Griffin, W.S.T., Stanley, L.C. , Ling, C , White, L. , MacLeod, V . , Perrot, L.J. , White, C.L. I l l Araoz, C. 1989. Brain interleukin 1 and S-100 immunoreactivity are elevated in Down syndrome and Alzheimer disease. PNAS 86:7611-7615. Griffin, W.S.T., Sheng, J.G., Roberts, G.W., Mrak R.E. 1995. IL-1 expression in different plaque types in Alzheimer's disease: significance in plaque evolution. J. Neuropathol. Exp. Neurol. 54:276-281. Griffin, W.S.T., Sheng, J.G., Royston, M.C. , Gentleman, S.M., McKenzie, J.E., Graham, D.I., Roberts, G.W., Mrak, R.E. 1998. Glial-neuronal interactions in Alzheimer's disease: the potential role of.a cytokine cycle in disease progression. Brain Pathol. 8:65-72. Grimaldi, M . , Atzori, M . , Ray, P., Alkon, D.L. Mobilization of calcium from intracellular stores, potentiation of neurotransmitter-induced calcium transients, and capacitative calcium entry by 4-aminopyridine. J. Neurosci, 21:3135-3143. Grissmer, S., Ghanshani, S., Dethlefs. B. , MacPherson, J.D., Wasmuth, J.J., Gutman, G.A., Cahalan, M.D. , Chandy, K . G . 1992. The Shaw-related potassium channel gene, Kv3.1, on human chromosome 11, encodes the type / K + channel in T cells. J. Biol . Chem. 267:20971-20979. Grissmer, S., Nguyen, A . N . , Aiyar, J., Hanson, D.C., Mather, R.J., Gutman, G.A., Karmilowicz, M.J. , Auperin, D.D., Chandy, K . G . 1994. Pharmacological characterization of five cloned voltage-gated K + channels, types K v l . l , 1.2, 1.3, 1.5, and 3.1, stably expressed in mammalian cell lines. Mol Pharmacol. 45:1227-1234. Guillemin, G.J., Smythe, G., Takikawa, O., Brew, B.J. 2005. Expression of indoleamine 2,3-dioxygenase and production of quinolinic acid by human microglia, astrocytes, and neurons. Glia. 49:15-23. Guillemin, G.J., Williams, K.R., Smith, D.G., Smythe, G.A., Croitoru-Lamoury, J., Brew, B.J. 2003. Quinolinic acid in the pathogenesis of Alzheimer's disease. Adv. Exp. Med. Biol . 527:167-176. Haga, S., Ikeda, K. , Sato, M . , Ishii, T. 1993. Synthetic Alzheimer amyloid beta/A4 peptides enhance production of complement C3 component by cultured microglial cells. Brain Res. 601:88-94. Hanisch, U .K. 2002. Microglia as a source and target of cytokines. Glia. 40:140-155. Haroutunian, V . , Barnes, E., Davis, K . L . 1985. Cholinergic modulation of memory in rats. Psychopharm. 87:266-271. Harrison, J.K., Jiang, Y . , Chen, S., Xia , Y . , Maciejewski, D., McNamara, R.K., Streit, W.J., Salafranca, M . N . , Adhikari, S., Thompson, D.A., Botti, P., Bacon, K .B . , Feng, L . 1998. Role for neuronally derived fractalkine in mediating interactions between neurons and CX3CR1-expressing microglia. PNAS 95: 10896-10901. Heese, K. , Hock, C , Otten, U . 1998. Inflammatory signals induce neurotrophin expression in human microglial cells. J. Neurochem. 70:699-707. Hensley, K. , Maidt, M . , Yu, Z., Sang, H. , Markesbery, W.R., Floyd, R.A. 1998. Electrochemical analysis of protein nitrotyrosine and dityrosine in the Alzheimer brain indicates region-specific accumulation. J. Neurosci. 18:8126-32. Hensley, K. , Floyd, R.A., Zheng, N . Y . , Nael, R., Robinson, K . A . , Nguyen, X . , Pye, Q.N., Stewart, C.A., Geddes, J., Markesbery, W.R., Patel, E., Johnson, G.V., Bing, G. 1999. p38 kinase is activated in the Alzheimer's disease brain. J. Neurochem. 72:2053-2058. Heyen, J.R., Ye, S., Finck, B.N. , Johnson, R.W. 2000. Interleukin (IL)-10 inhibits IL-6 production in microglia by preventing activation of NF-kappaB. Brain Res. Mol . Brain Res. 77:138-147. Hide, I., Tanaka, M . , Inoue, A. , Nakajima, K. , Kohsaka, S., Inoue, K. , Nakata, Y . 2000. Extracellular ATP triggers tumor necrosis factor-alpha release from rat microglia. J. Neurochem. 75:965-972. Higgins, G.A., Jacobsen, H . 2003. Transgenic mouse models of Alzheimer's disease: phenotype and application. Behav. Pharmacol. 14:419—438 Ho, L. , Luterman, J.D., Aisen, P.S., Pasinetti, G .M. , Montine, T.J., Morrow, J.D. 2000. Elevated CSF prostaglandin E 2 levels in patients with probable A D . Neurology 55:323. Ho, L. , Pieroni, C , Winger, D., Purhoit, D.P., Aisen, P.S., Pasinetti, G . M . 1999. Regional distribution of cyclooxygenase-2 in the hippocampal formation in Alzheimer's disease. J. Neurosci. Res. 57, 295-303. Hock, C , Konietzko, U . , Papassotiropoulos, A. , Wollmer, A. , Streffer, J., von Rotz, R.C., Davey, G., Moritz, E., Nitsch, R . M . 2002. Generation of antibodies specific for beta- amyloid by vaccination of patients with Alzheimer disease. Nat. Med. 8:1270-1275. Hock, C , Konietzko, U . , Streffer, J.R., Tracy, J., Signorell, A . , Muller-Tillmanns, B. , Lemke, U . , Henke, K. , Moritz, E., Garcia, E., Wollmer, M.A . , Umbricht, D., de Quervain, D.J., Hofmann, M . , Maddalena, A . , Papassotiropoulos, A. , Nitsch, R . M . 2003. Antibodies against beta-amyloid slow cognitive decline in Alzheimer's disease. Neuron. 38:547-554. Hoozemans, J.J., Bruckner, M . K . , Rozemuller, A.J . , Veerhuis, R., Eikelenboom, P., Arendt, T. 2002a. Cyclin D l and cyclin E are co-localized with cyclo-oxygenase 2 (COX-2) in pyramidal neurons in Alzheimer disease temporal cortex. J. Neuropathol. Exp. Neurol. 61:678-88 Hoozemans, J.J.M., Rozemuller, A . J .M. , Janssen, I., DeGroot, C.J.A., Veerhuis, R., Eikelenboom, P. 2001. Cyclooxygenase expression in microglia and neurons in Alzheimer's disease and control brain. Acta Neuropathol. 101, 2-8. Hoozemans, J.J.M., Veerhuis, R., Janssen, I., van Elk, E., Rozemuller, A . J .M. , Eikelenboom, P. 2002b. The role of cyclo-oxygenase 1 and 2 activity in prostaglandin E2 secretion by cultured human adult microglia: Implications for Alzheimer's Disease. Brain Res. 951,218-226. Hu, P. and Fredholm, B. B. 1991. 4-Aminopyridine-induced increase in basal and stimulation-evoked [ 3H]-NA release in slices from rat hippocampus: C a 2 + sensitivity and presynaptic control. Br. J. Pharmacol. 102: 764-768. Hull, M . , Berger, M . , Volk, B. , Bauer, J. 1996. Occurrence of IL-6 in cortical plaques of Alzheimer's disease patients may precede transformation of diffuse into neuritic plaques. Ann. N . Y . Acad. Sci. 777:205-212. Husemann, J., Loike, J.D., Anankov, R , Febbraio, M . , Silverstein, S.C. 2002. Scavenger receptors in neurobiology and neuropathology: their role on microglia and other cells of the nervous system. Glia. 40:195-205. Husemann, J., Loike, J.D., Kodama, T., Silverstein, S.C. 2001. Scavenger receptor class B type I (SR-BI) mediates adhesion of neonatal murine microglia to fibrillar beta- amyloid. J. Neuroimmunol. 114:142-150. Illes, P., Norenberg, W., Gebicke-Haerter, P.J. 1996. Molecular mechanisms of microglial activation. B . Voltage- and purinoceptor-operated channels in microglia. Neurochem Int. 29:13-24. Ilschner, S., Ohlemeyer, C , Gimpl, G., Kettenmann, H . 1995. Modulation of potassium currents in cultured murine microglial cells by receptor activation and intracellular pathways. Neuroscience. 66:983-1000. Ilschner, S., Nolte, C , Kettenmann, H . 1996. Complement factor C5a and epidermal growth factor trigger the activation of outward potassium currents in cultured murine microglia. Neurosci. 73:1109-1120 in t' Veld, B.A. , Ruitenberg, A . , Hofman, A. , Launer, L.J. , van Duijn, C M . , Stijnen, T., Breteler, M . M . , Strieker, B .H. 2001. Nonsteroidal antiinflammatory drugs and the risk of Alzheimer's disease. N . Engl. J. Med. 345:1515-1521. Ishizuka, K. , Kimura, T., Igata-Yi, R., Katsuragi, S., Takamatsu, J., Miyakawa, T., 1997. Identification of monocyte chemoattractant protein-1 in senile plaques and reactive microglia of Alzheimer's disease. Psychiatry Clin Neurosci 51:135-138. Itagaki, S., McGeer, P.L., Akiyama, H. , Zhu, S., Selkoe, D. 1989. Relationship of microglia and astrocytes to amyloid deposits of Alzheimer disease. J Neuroimmunol. 24:173-182. IUIS/WHO Subcommittee on Chemokine Nomenclature. 2003. Chemokine/chemokine receptor nomenclature. Cytokine 21:48-49. Janabi, N . , D i Stefano, M . , Wallon, C , Hery, C , Chiodi, F., Tardieu, M . 1998. Induction of human immunodeficiency virus type 1 replication in human glial cells after proinflammatory cytokines stimulation: effect of IFNgamma, ILlbeta, and TNFalpha on differentiation and chemokine production in glial cells. Glia. 23:304-315. Jankowska, E., Lundberg, A . , Rudomin, P., Sykova, E. 1982. Effects of 4-aminopyridine on synaptic transmission in the cat spinal cord. Brain Res. 240:117-129. Jiang, B. , Sun, X . , Cao, K. , Wang, R. 2002. Endogenous K v channels in human embryonic kidney (HEK-293) cells. Mol Cell Biochem. 238:69-79. Jorm, A.F. , Jolley, D. 1998. The incidence of dementia: a meta-analysis. Neurology. 51:728-733. Kagan, B.L. , Hirakura, Y . , Azimov, R., Azimova, R., Lin, M.C. 2002. The channel hypothesis of Alzheimer's disease: current status. Peptides. 23:1311-1315. Kang, J., Park, E.J., Jou, I., Kim, J.H., Joe, E.H. 2001. Reactive oxygen species mediate Abeta(25-35)-induced activation of BV-2 microglia. Neuroreport. 12:1449-1452. Kaur, C , Hao, A.J . , Wu, C.H., Ling, E.A. 2001. Origin of microglia. Microsc. Res. Tech. 54:2-9. Kawahara, M . , Kuroda, Y . 2000. Molecular mechanism of neurodegeneration induced by Alzheimer's beta-amyloid protein: channel formation and disruption of calcium homeostasis. Brain Res. Bull. 53:389-397. Kawas, C , Gray, S., Brookmeyer, R , Fozard, J., Zonderman, A . 2000. Age-specific incidence rates of Alzheimer's disease: the Baltimore Longitudinal Study of Aging. Neurology. 54:2072-2077. Kettenmann H , Hoppe D, Gottmann K , Banati R, Kreutzberg G. Cultured microglial cells have a distinct pattern of membrane channels different from peritoneal macrophages. J Neurosci Res. 1990 Jul;26(3):278-87. Kettenmann H , Banati R, Walz W. Electrophysiological behavior of microglia. Glia. 1993 Jan;7(l):93-101. Khanna, R., Roy, L. , Zhu, X . , Schlichter, L .C. 2001. K+ channels and the microglial respiratory burst. A m J Physiol Cell Physiol. 280:C796-806. Kim, S.H., Smith, C.J., Van Eldik, L.J. 2004. Importance of M A P K pathways for microglial pro-inflammatory cytokine IL-1 beta production. Neurobiol Aging. 25:431- 439. Kish, S.J., Bergeron, C , Rajput, A . , Dozic, S., Mastrogiacomo, F., Chang, L.J. , Wilson, J .M., DiStefano, L . M . , Nbbrega, J.N. 1992. Brain cytochrome oxidase in Alzheimer's disease. J Neurochem. 59:776-779. Kitamura, Y . , Taniguchi, T., Kimura, H. , Nomura, Y . , Gebicke-Haerter, P.J. 2000. lnterleukin-4-inhibited mRNA expression in mixed rat glial and in isolated microglial cultures. J. Neuroimmunol. 106:95-104. Klee, R., Heinemann, U . , Eder, C. 1999. Voltage-gated proton currents in microglia of distinct morphology and functional state. Neuroscience. 91:1415-1424. Klegeris, A . and McGeer, P.L. 1994. Rat brain microglia and peritoneal macrophages show similar responses to respiratory burst stimulants. J. Neuroimmunol. 53:83-90. Klegeris, A . , McGeer, P.L. 1997. beta-amyloid protein enhances macrophage production of oxygen free radicals and glutamate. J. Neurosci. Res. 49:229-235. Klegeris A , McGeer EG, McGeer PL. 2000. Inhibitory action of l-(2-chlorophenyl)-N- methyl-N-(l-methylpropyl)-3-isoquinolinecarboxam ide (PK 11195) on some mononuclear phagocyte functions. Biochem Pharmacol. 59:1305-1314. Klegeris, A . , Walker, D.G., McGeer, P.L. 1999. Toxicity of human THP-1 monocytic cells towards neuron-like cells is reduced by non-steroidal anti-inflammatory drugs (NSAIDs). Neuropharmacology. 38:1017-1025. Koh, J.Y., Yang, L .L . , Cotman, C.W. 1990. Beta-amyloid protein increases the vulnerability of cultured cortical neurons to excitotoxic damage. Brain Res. 533:315-320. Koistinaho, M and Koistinaho, J. 2002. Role of p38 and p44/42 mitogen-activated protein kinases in microglia. Glia 40:175-183. Korotzer, A.R., Whittemore, E.R., Cotman, C.W. 1995. Differential regulation by beta- amyloid peptides of intracellular free Ca2+ concentration in cultured rat microglia. Eur. J. Pharmacol. 288:125-130. Kotecha, S.A., Schlichter, L .C. 1999. A K v l . 5 to Kv l .3 switch in endogenous hippocampal microglia and a role in proliferation. J Neurosci. 19:10680-10693. Kotilinek, L .A. , Bacskai, B. , Westerman, M . , Kawarabayashi, T., Younkin, L. , Hyman, B.T., Younkin, S., Ashe, K . H . 2002. Reversible memory loss in a mouse transgenic model of Alzheimer's disease. J Neurosci. 22:6331-6335. Kowall, N.W., Beal, M.F. , Busciglio, J., Duffy, L .K. , Yankner, B .A. 1991. A n in vivo model for the neurodegenerative effects of beta amyloid and protection by substance P. Proc Natl Acad Sci U S A . 88:7247-7251. Kreutzberg, G.W. 1996. Microglia: a sensor for pathological events in the CNS. Trends Neurosci. 19:312-318. Lajdova, I., Chorvat Jr, D., Spustova, V . , Chorvatova, A . 2004. 4-Aminopyridine activates calcium influx through modulation of the pore-forming purinergic receptor in human peripheral blood mononuclear cells. Can J Physiol Pharmacol. 82:50-56. Landreth, G.E., Heneka, M.T. 2001. Anti-inflammatory actions of peroxisome proliferator-activated receptor gamma agonists in Alzheimer's disease. Neurobiol Aging. 22:937-944. Lee, S. C , Dickson, D. W., Brosnan, C. F. 1995. Interleukin-1, nitric oxide and reactive astrocytes. Brain Behav. Immun. 9:345-354. Lee, S.C., Liu, W., Dickson, D.W., Brosnan, C.F., Berman, J.W. 1993. Cytokine production by human fetal microglia and astrocytes. J. Immunol. 150, 2659-2667. Lee, Y . B . , Nagai, A . , Kim, S.U. 2002. Cytokines, chemokines, and cytokine receptors in human microglia. J. Neurosci. Res. 69:94-103. Lee, Y . B . , Schrader, J.W., Kim, S.U. 2000. p38 map kinase regulates TNF-alpha production in human astrocytes and microglia by multiple mechanisms. Cytokine. 12:874-880. Lemeignan, M . , Millart, H. , Lamiable, D., Molgo, J., Lechat, P. 1984. Evaluation of 4- aminopyridine and 3,4-diaminopyridine penetrability into cerebrospinal fluid in anesthetized rats. Brain Res. 304:166-169. Levy-Lahad, E., Wasco, W., Poorkaj, P., Romano, D . M . , Oshima, J., Pettingell, W.H., Yu , C.E., Jondro, P.D., Schmidt, S.D., Wang, K . 1995. Candidate gene for the chromosome 1 familial Alzheimer's disease locus. Science 269:973-977. L i , L. , Bressler, B., Prameya, R., Dorovini-Zis, K. , Van Breemen, C. 1999. Agonist- stimulated calcium entry in primary cultures of human cerebral microvascular endothelial cells. Microvasc Res. 57:211-26. L i , Y . , Liu, L. , Kang, J., Sheng, J.G., Barger, S.W., Mrak, R.E., Griffin, W.S. 2000. Neuronal-glial interactions mediated by interleukin-1 enhance neuronal acetylcholinesterase activity and mRNA expression. J. Neurosci. 20:149-155. Ling, E., Wong, W. 1993. The origin and nature of ramified and ameboid microglia: a historical review and current concepts. Glia 7:9-18. Ling, Y . , Morgan, K. , Kalsheker, N . 2003. Amyloid precursor protein (APP) and the biology of proteolytic processing: relevance to Alzheimer's disease. Int J Biochem Cell Biol. 35:1505-1535. Lorton, D. 1997. beta-Amyloid-induced IL-1 beta release from an activated human monocyte cell line is calcium- and G-protein-dependent. Mech Ageing Dev. 94:199-211. Lorton, D., Schaller, J., Lala, A . , De Nardin, E. 2000. Chemotactic-like receptors and Abeta peptide induced responses in Alzheimer's disease. Neurobiol Aging. 21:463-473. Lue, L.F., Kuo, Y . M . , Roher, A.E . , Brachova, L. , Shen, Y . , Sue, L. , Beach, T., Kurth, J.H., Rydel, R.E., Rogers, J. 1999. Soluble amyloid beta peptide concentration as a predictor of synaptic change in Alzheimer's disease. A m J Pathol. 155:853-862. Lue, L.F., Rydel, R., Brigham, E.F., Yang, L .B. , Hampel, H. , Murphy, G.M. , Brachova, L. , yan, S.D., Walker, D.G., Shen, Y . , Rogers J. 2001a. Inflammatory repertoire of Alzheimer's disease and nondemented elderly microglia in vitro. Glia 35:72-79. Lue, L.F., Walker, D.G. 2002. Modeling Alzheimer's disease immune therapy mechanisms: interactions of human postmortem microglia with antibody-opsonized amyloid beta peptide. J Neurosci Res. 70:599-610. Lue, L.F., Walker, D.G., Brachova, L. , Beach, T.G., Rogers, J., Schmidt, A . M . , Stern, D .M. , Yan, S.D. 2001b. Involvement of microglial receptor for advanced glycation endproducts (RAGE) in Alzheimer's disease: identification of a cellular activation mechanism. Exp Neurol. 171:29-45. Lustbader, J.W., Chi l l i , M . , Lin, C , Xu , H.W., Takuma, K. , Wang, N . , Caspersen, C , Chen, X . , Pollak, S., Chaney, M . , Trinchese, F., Liu, S., Gunn-Moore, F., Lue, L.F., Walker, D.G., Kuppusamy, P., Zewier, Z.L. , Arancio, O., Stern, D., Yan, S.S., Wu, H . 2004. A B A D directly links Abeta to mitochondrial toxicity in Alzheimer's disease. Science. 304:448-452. Maciejewski-Lenoir, D., Chen, S., Feng, L. , Maki, R., Bacon, K . B . 1999. Characterization of fractalkine in rat brain cells: migratory and activation signals for CX3CR-1-expressing microglia. J Immunol. 163:1628-1635. Mackenzie, I.R., Hao, C , Munoz, D.G. 1995. Role of microglia in senile plaque formation. Neurobiol. Aging 16:797-804 Mackenzie, I.R., Munoz, D.G. 1998. Nonsteroidal anti-inflammatory drug use and Alzheimer-type pathology in aging. Neurology. 50:986-990. Madrid, L .V . , Mayo, M.W., Reuther, J.Y., Baldwin, A.S. Jr. 2001. Akt stimulates the transactivation potential of the RelA/p65 Subunit of NF-kappa B through utilization of the Ikappa B kinase and activation of the mitogen-activated protein kinase p38. J. Biol . Chem. 276:18934-18940. Mahley, R.W. 1988. Apolipoprotein E: cholesterol transport protein with expanding role in cell biology. Science 240:622-630. Mahley R.W. and Rail Jr., S.C. 2000. Apolipoprotein E: far more than a lipid transport protein. Ann. Rev. Genomics Hum. Genet. 1:507-537. Mark, R.J., Hensley, K. , Butterfield, D.A., Mattson, M.P. 1995. Amyloid beta-peptide impairs ion-motive ATPase activities: evidence for a role in loss of neuronal Ca2+ homeostasis and cell death. J Neurosci. 15:6239-6249. Mark, R.J., Keller, J.N., Kruman, I., Mattson, M.P. 1997. Basic FGF attenuates amyloid beta-peptide-induced oxidative stress, mitochondrial dysfunction, and impairment of Na+/K+-ATPase activity in hippocampal neurons. Brain Res. 756:205-214. Martinez, F.O., Sironi, M . , Vecchi, A . , Colotta, F., Mantovani, A. , Locati, M . 2004. IL-8 induces a specific transcriptional profile in human neutrophils: synergism with LPS for IL-1 production. Eur. J. Immunol. 34, 2286-2292. Masters, C. L. , Simms, G., Weinman, N .A. , Multhaup, G., McDonald, B.L. , Beyreuther, K . 1985. Amyloid plaque core protein in Alzheimer disease and Down syndrome. PNAS 82:4245^1249. Matter, M.L . , Zhang, Z., Nordstedt, C , Ruoslahti, E. 1998. The alpha5betal integrin mediates elimination of amyloid-beta peptide and protects against apoptosis. J Cell Biol . 141:1019-1030. Mattson, M.P. 2002. Oxidative stress, perturbed calcium homeostasis, and immune dysfunction in Alzheimer's disease. J. Neurovirol. 8:539-550. Mattson, M.P., Chan, S.L. 2003. Neuronal and glial calcium signaling in Alzheimer's disease. Cell Calcium. 34:385-397. Mattson, M.P., Cheng, B. , Davis, D., Bryant, K. , Lieberburg, I., Rydel, R.E. 1992. beta- Amyloid peptides destabilize calcium homeostasis and render human cortical neurons vulnerable to excitotoxicity. J Neurosci. 12:376-389. McCloskey, M.A . , Cahalan, M.D. 1990. G protein control of potassium channel activity in a mast cell line. J Gen Physiol. 95:205-227. McDonald, D.R., Bamberger, M.E. , Combs, C.K., Landreth, G.E. 1998. beta-Amyloid fibrils activate parallel mitogen-activated protein kinase pathways in microglia and THP1 monocytes. J. Neurosci. 18:4451-4460. McDonald, D.R., Brunden, K.R., Landreth, G.E. 1997. Amyloid fibrils activate tyrosine kinase-dependent signaling and superoxide production in microglia. J. Neurosci. 17, 2284-2294. McGeer, P.L., Itagaki, S., Tago, FL, McGeer, E.G. 1988. Occurrence of H L A - D R reactive microglia in Alzheimer's disease. Ann N Y Acad Sci. 540:319-23. McGeer, P.L. and McGeer, E.G. 1995. The inflammatory response system of brain:implications for therapy of Alzheimer and other neurodegenerative diseases. Brain Res. Rev. 21:195-218. McGeer, P.L. and McGeer, E.G. 1999. Inflammation of the brain in Alzheimer's disease: implications for therapy. J. Leukoc. Biol. 65:409-415. McGeer, P.L. and McGeer, E.G. 2001. Polymorphisms in inflammatory genes and the risk of Alzheimer disease. Arch Neurol. 58:1790-1792. McGeer, P L . , McGeer, E.G., Yasojima, K . 2000. Alzheimer disease and neuroinflammation. J. Neural Trans. Suppl. 59:53-57. McGeer, P L . , Schulzer, M . , McGeer, E.G. 1996. Arthritis and anti-inflammatory agents as possible protective factors for Alzheimer's disease: a review of 17 epidemiologic studies. Neurology. 47:425-432. McKee, A .C . , Kosik, K.S. , Kowall, N.W. 1991. Neuritic pathology and dementia in Alzheimer's disease. Ann. Neurol. 30:156-165. McKinney, L .C. and Gallin, E .K. 1993. G-protein activators induce a potassium conductance in murine macrophages. J. Memb. Biol . 130:265-76. McLarnon, J.G., Xu , R., Lee, Y .B . , Kim, S.U. 1997. Ion channels of human microglia in culture. Neuroscience. 78:1217-1228. McLarnon, J.G., Zhang, L. , Goghari, V . , Lee, Y . B . , Walz, W., Krieger, C , Kim, S.U. 1999. Effects of ATP and elevated K + on K + currents and intracellular C a 2 + in human microglia. Neuroscience. 91:343-352. McManus, C M . , Brosnan, C.F, Berman, J.W. 1998. Cytokine induction of MIP-1 alpha and MIP-1 beta in human fetal microglia. J Immunol. 160:1449-1455. Meda, L., Baron, P., Prat, E., Scarpini, E., Scarlato, G., Cassatella, M.A . , Rossi F. 1999. Proinflammatory profile of cytokine production by human monocytes and murine microglia stimulated with P-amyloid (25-35). J Neuroimmunol 93: 45-52. Meda, L. , Baron, P., Scarlato, G. 2001. Glial activation in Alzheimer's disease: the role of Ap and its associated proteins. Neurobiol. Aging 22:885-893. Meda, L. , Bernasconi, S., Bonaiuto, C , Sozzani, S., Zhou, D., Otvos, L. , Mantovani, A. , Rossi, F., Cassatella, M . A . 1996. P-amyloid (25-35) peptide, and EFNy synergistically induce the production of the chemotactic cytokine M C P - l / J E in monocytes, and microglial cells. J Immunol 157:1213-1218. Meda, L. , Cassatella, M.A . , Szendrei, G., Otvos, L. , Baron, P., Villalba, M . , Ferrari, D., Rossi, F. 1995. Activation of microglial cells by P-amyloid protein and IFNy. Nature 374 :647-650 Mehlhorn, G., Hollborn, M . , Schliebs, R. 2000. Induction of cytokines in glial cells surrounding cortical beta-amyloid plaques in transgenic Tg2576 mice with Alzheimer pathology. Int. J. Dev. Neurosci. 18:423-431. Mellman, I., Koch, T., Healey, G., Hunziker, W., Lewis, V . , Plutner, H. , Miettinen, H . , Vaux, D., Moore, K. , Stuart, S. 1988. Structure and function of Fc receptors on macrophages and lymphocytes. J Cell Sci Suppl. 9:45-65. Merrill, J.E., Ignarro, L.J. , Sherman, M P . , Melinek, J., Lane, T.E. 1993. Microglial cell cytotoxicity of oligodendrocytes is mediated through nitric oxide. J Immunol. 151:2132- 2141. Minghetti, L. , Levi, G. 1998. Microglia as effector cells in brain damage and repair: Focus on prostanoids and nitric oxide. Prog Neurobiol 54:99-125. Mitrasinovic, O.M. and Murphy Jr., G . M . 2003. Microglial overexpression of the M-CSF receptor augments phagocytosis of opsonized Ap . Neurobiol. Aging. 24: 807-815. Montine, T.J., Sidell, K.R., Crews, B.C., Markesbery, W.R., Marnett, L.J. , Roberts, L.J. , Morrow, J.D. 1999. Elevated CSF prostaglandin E 2 levels in patients with probable A D . Neurology 53:1495-1498. Moore, K.J . , E l Khoury, J., Medeiros, L .A . , Terada, K. , Geula, C , Luster, A .D. , Freeman, M.W. 2002. A CD36-initiated signaling cascade mediates inflammatory effects of beta-amyloid. J Biol Chem. 277:47373-47379. Morihata, H. , Nakamura, F., Tsutada, T., Kuno, M . 2000. Potentiation of a voltage-gated proton current in acidosis-induced swelling of rat microglia. J. Neurosci. 20:7220-7227. Mrak, R.E. and Griffin, W.S. 2000. IL-1, and the immunogenetics of Alzheimer's disease. J. Neuropath. Exp. Neurol. 59:471—476. Murphy, A . , Sunohara, J.R., Sundaram, M . , Ridgway, N.D. , McMaster, C.R., Cook, H.W., Byers, D . M . 2003. Induction of protein kinase C substrates, Myristoylated alanine- rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP), by amyloid beta-protein in mouse BV-2 microglial cells. Neurosci Lett. 347:9-12. Murphy, G . M . Jr., Yang, L . , Cordell, B. 1998. Macrophage colony-stimulating factor augments beta-amyloid-induced interleukin-1, interleukin-6, and nitric oxide production by microglial cells. J. Biol. Chem. 273, 20967-20971. Mutisya, E .M. , Bowling, A .C . , Beal, M.F. 1994. Cortical cytochrome oxidase activity is reduced in Alzheimer's disease. J Neurochem. 63:2179-2184. Nagai, A . , Nakagawa, E., Hatori, K. , Choi, H.B., McLarnon, J.G., Lee, M . A . and Kim, S.U. 2001. Generation and characterization of immortalized human microglial cell lines: expression of cytokines and chemokines, Neurobiol. Dis. 8, 1057-1068. Nakai, M . , Tanimukai, S., Yagi, K. , Saito, N . , Taniguchi, T., Terashima, A. , Kawamata, T., Yamamoto, H. , Fukunaga, K. , Miyamoto, E., Tanaka, C. 2001. Amyloid beta protein activates PKC-delta and induces translocation of myristoylated alanine-rich C kinase substrate (MARCKS) in microglia. Neurochem Int. 38:593-600. Nakajima, K. , Kikuchi, Y . , Ikoma, E., Honda, S., Ishikawa, M . , Liu, Y . , Kohsaka, S. 1998. Neurotrophins regulate the function of cultured microglia. Glia. 24:272-289. Nakanishi, H . 2003. Neuronal and microglial cathepsins in aging and age-related diseases. Ageing Res Rev. 2:367-81. Narita, M . , Holtzman, D .M. , Schwartz, A . L . , Bu, G. 1997. Alpha2-macroglobulin complexes with and mediates the endocytosis of beta-amyloid peptide via cell surface low-density lipoprotein receptor-related protein. J Neurochem 69:1904-1911 Naslund, J., Haroutunian, V . , Mohs, R., Davis,' K .L . , Davies, P., Greengard, P., Buxbaum, J.D. 2000. Correlation between elevated levels of amyloid beta-peptide in the brain and cognitive decline. J. Am. Med. Assoc. 283:1571-1577. Nilson, L .N . , Bales, K.R., DiCarlo, G., Gordon, M . N . , Morgan, D., Paul, S.M., Potter, H . 2001. Alpha-1-antichymotrypsin promotes beta-sheet amyloid plaque deposition in a transgenic mouse model of Alzheimer's disease. J Neurosci. 21:1444-1451. Nishiyori, A . , Minami, M . , Ohtani, Y . , Takami, S., Yamamoto, J., Kawaguchi, N . , Kume, T., Akaike, A . , Satoh, M . 1998. Localization of fractalkine and CX3CR1 mRNAs in rat brain: does fractalkine play a role in signaling from neuron to microglia? FEBS Lett. 429,: 167-172. Nordberg, A . 1992. Neuroreceptor changes in Alzheimer disease. Cerebrovasc. Brain Met. Rev. 4:303-328 Norenberg, W., Appel, K. , Bauer, J., Gebicke-Haerter, P.J., Illes, P. 1993. Expression of an outwardly rectifying K + channel in rat microglia cultivated on teflon. Neurosci. Lett. 160: 69-72. Norenberg, W., Gebicke-Haerter, P.J., Illes, P. 1992. Inflammatory stimuli induce a new K+ outward current in cultured rat microglia. Neurosci Lett. 147:171 -174. Norenberg, W., Gebicke-Haerter, P.J., Illes, P. 1994. Voltage-dependent. potassium channels in activated rat microglia. J Physiol. 475:15-32. Oddo, S., Caccamo, A. , Shepherd, J.D., Murphy, M P . , Golde, T.E., Kayed, R., Metherate, R., Mattson, M.P., Akbari, Y . , LaFerla, F . M . 2003. Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Abeta and synaptic dysfunction. Neuron. 39:409-421. Okamoto, T., Takeda, S., Murayama, Y . , Ogata, E., Nishimoto, I. 1995. Ligand- dependent G protein coupling function of amyloid transmembrane precursor. J. Biol. Chem. 270:4205^1208. Oltersdorf, T., Ward, P.J., Henriksson, T., Beattie, E.C., Neve, R., Lieberburg, I., Fritz, L .C. 1990. The Alzheimer amyloid precursor protein. Identification of a stable intermediate in the biosynthetic/degradative pathway. J Biol Chem. 265:4492-4497. O'Neill , G.P., Ford-Hutchinson, A.W. 1993. Expression of mRNA for cyclooxygenase-1 and cyclooxygenase-2 in human tissues. FEBS Lett. 330,156-160. O'Neill , L .A . and Kaltschmidt, C. 1997. NF-kappaB: a crucial transcription factor for glial and neuronal cell function. Trends Neurosci. 20:252-258. Paganini-Hill, A . and Henderson, V .W. 1994. Estrogen deficiency and risk of Alzheimer's disease in women. Am. J. Epidemiol. 140:256-261. Pasinetti, G . M . and Aisen, P.S. 1998. Cyclooxygenase-2 expression is increased in frontal cortex of Alzheimer's disease brain. Neuroscience 87:319-24. Paxinos, G. and Watson, C. The rat brain in stereotaxic coordinates. Ed. 2. New York: Academic. 1986. Peress, N.S., Fleit, H.B., Perillo, E., Kuljis, R., Pezzullo, C. 1993. Identification of Fc gamma RI, II and III on normal human brain ramified microglia and on microglia in senile plaques in Alzheimer's disease. J Neuroimmunol. 48:71-79. Perez, R.G., Zheng, H. , Van der Ploeg, L.H.T., Koo, E.H. 1997. The P-amyloid precursor protein of Alzheimer's disease enhances neuron viability and modulates neuronal polarity. J. Neurosci. 17:9407-9414. Pericak-Vance, M.A . , Bebout, J.L., Gaskell Jr., P.C., Yamaoka, L .H . , Hung, W.Y., Alberts, M.J. , Walker, A.P., Bartlett, R.J., Haynes, C.A., Welsh, K . A . 1991. Linkage studies in familial Alzheimer disease: evidence for chromosome 19 linkage. A m J Hum Genet 48:1034-1050. Pericak-Vance, M.A . , Bass, M.P., Yamaoka, L .H . , Gaskell, P.C., Scott, W.K., Terwedow, H.A., Menold, M . M . , Conneally, P .M. , Small, G.W., Vance, J .M., Saunders, A . M . , Roses, A.D. , Haines, J.L. 1997. Complete genomic screen in late-onset familial Alzheimer disease. Evidence for a new locus on chromosome 12. J A M A 278:1237-1241. Perney, T.M. , L i , X . , Kang, S., Kaczmarek, L .K. , Birnberg, N . 1992. Regulation of mRNA for the Kv3.1 potassium channel in vitro and in vivo. Soc. Neurosci.Abstr. 18:1093. Perry, V . H . , Hume, D.A., Gordon, S. 1985. Immunohistochemical localization of macrophages and microglia in the adult and developing mouse brain. Neuroscience. 15:313-326. Peterson, P.K., Hu, S., Salak-Johnson, J., Molitor, T.W., Chao, C.C. 1997. Differential production of and migratory response to beta chemokines by human microglia and astrocytes. J. Infectious Dis. 175:478-481. Petit, A . , Bihel, F., Alves da Costa, C , Pourquie, O., Checler, F., Kraus, J.L. 2001. New protease inhibitors prevent gamma-secretase-mediated production of Abeta40/42 without affecting Notch cleavage. Nat Cell Biol . 3:507-511. Piani, D., Spranger, M . , Frei, K. , Schaffner, A . , Fontana, A . 1992. Macrophage-induced cytotoxicity of N-methyl-D-aspartate receptor positive neurons involves excitatory amino acids rather than reactive oxygen intermediates and cytokines. Eur. J. Immunol. 22:2429- 2436 Pierrot, N . , Ghisdal, P., Caumont, A.S., Octave, J.N. 2004. Intraneuronal amyloid-betal- 42 production triggered by sustained increase of cytosolic calcium concentration induces neuronal death. J Neurochem. 88:1140-1150. Pike, C.J., Burdick, D., Walencewicz, A.J . , Glabe, C.G., Cotman, C.W. 1993. Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state. J Neurosci. 13:1676-1687. Poduslo, J.F., Curran, G.L., Wengenack, T .M. , Malester, B. , Duff, K . 2001. Permeability of proteins at the blood-brain barrier in the normal adult mouse and double transgenic mouse model of Alzheimer's disease. Neurobiol Dis. 8:555-567. Puglielli, L. , Konopka, G., Pack-Chung, E., Ingano, L . A . , Berezovska, O., Hyman, B. T., Chang, T. Y . , Tanzi, R. E., Kovacs, D. M . 2001. Acyl-coenzyme A : cholesterol acyltransferase modulates the generation of the amyloid beta-peptide. Nat. Cell Biol . 3, 905-912. Pyo, H. , Jou, I., Jung, S., Hong, S., Joe, E.H. 1998. Mitogen-activated protein kinases activated by lipopolysaccharide and beta-amyloid in cultured rat microglia. Neuroreport 9:871-874. Qu, B. , Rosenberg, R.N. , L i , L. , Boyer, P.J., Johnston, S.A. 2004. Gene vaccination to bias the immune response to amyloid-beta peptide as therapy for Alzheimer disease. Arch Neurol. 61:1859-1864. Raivich, G., Bohatschek, M . , Kloss, C.U., Werner, A . , Jones, L .L . , Kreutzberg, G.W. 1999. Neuroglial activation repertoire in the injured brain: graded response, molecular mechanisms and cues to physiological function. Brain Res Brain Res Rev. 30:77-105. Raivich, G., Bluethmann, H. , Kreutzberg, G.W. 1996. Signaling molecules and neuroglial activation in the injured central nervous system. Keio J Med. 45:239-47. Refolo, L . M . , Pappolla, M.A . , LaFrancois, J., Malester, B. , Schmidt, S.D., Thomas- Bryant, T., Tint, G.S., Wang, R., Mercken, M . , Petanceska, S.S., Duff, K . E , 2001. A cholesterol-lowering drug reduces beta-amyloid pathology in a transgenic mouse model of Alzheimer's disease. Neurobiol. Dis. 8:890-899. Refolo, L . M . , Malester, B. , LaFrancois, J., Bryant-Thomas, T, Wang, R., Tint, G.S., Sambamurti, K. , Duff, K. , Pappolla, M . A . 2000. Hypercholesterolemia accelerates the Alzheimer's amyloid pathology in a transgenic mouse model. Neurobiol. Dis. 7:321-331. Richards, J.G., Higgins, G.A., Ouagazzal, A . M . , Ozmen, L. , Kew, J.N., Bohrmann, B. , Malherbe, P., Brockhaus, M . , Loetscher, H. , Czech, C , Huber, G., Bluethmann, H . , Jacobsen, H. , Kemp, J.A. 2003. PS2APP transgenic mice, coexpressing hPS2mut and hAPPswe, show age-related cognitive deficits associated with discrete brain amyloid deposition and inflammation. J Neurosci 23:8989-9003 Rogers, J., Cooper, N.R., Webster, S., Schultz, J., McGeer, P.L., Styren, S.D., Civin, W.H., Brachova, L. , Bradt, B. , Ward, P. et al. 1992. Complement activation by beta- amyloid in Alzheimer disease. PNAS 89:10016-10020. Rogers, J., Luber Narod, J., Styren, S.D. and Civin, W.H., 1988. Expression of immune system-associated antigens by cells of the human central nervous system: Relationship to the pathology of Alzheimer's disease. Neurobiol. Aging 9, pp. 339-349. Rogers, J., Strohmeyer, R , Kovelowski, C.J., L i , R. 2002. Microglia and inflammatory mechanisms in the clearance of amyloid beta peptide. Glia. 40:260-269. Roses, A .D . 1998. Alzheimer diseases: a model of gene mutations and susceptibility polymorphisms for complex psychiatric diseases. A m J Med Genet. 81:49-57. Sabo, S.L., Ikin, A.F. , Buxbaum, J.D. Greengard, P. 2001. The Alzheimer amyloid precursor protein (APP) and FE65, an APP-binding protein, regulate cell movement. J. Cell Biol. 153:1403-1414. Sakai, J., Hoshino, A. , Takahashi, S., Miura, Y . , Ishii, H. , Suzuki, H. , Kawarabayasi, Y . , Yamamoto, T. 1994. Structure, chromosome location; and expression of the human very low density lipoprotein receptor gene. J. Biol. Chem. 269:2173-2182. Santamaria, A . , Jimenez-Capdeville, M . E., Camacho, A . , Rodriguez-Martinez, E., Flores, A . Galvan-Arzate, S. 2001. In vivo hydroxyl radical formation after quinolinic acid infusion into rat corpus striatum. Neuroreport 12, 2693-2696 Sasaki, A . , Yamaguchi, H. , Ogawa, A . , Sugihara, S., and Nakazato, Y . 1997. Microglial activation in early stages of amyloid beta protein deposition. Acta Neuropathol. 94:316- 322. Satoh, J.I., Lee, Y . B . and Kim, S.U. 1995. T cell costimulatory molecules B7-11 (CD-80) and B7-2 (CD-86) are expressed in human microglia but not astrocytes in culture. Brain Res. 704, 92-96. Sawada, M . , Suzumura, A . , Hosoya, H. , Marunouchi, T., Nagatsu, T. 1999. Interleukin- 10 inhibits both production of cytokines and expression of cytokine receptors in microglia. J Neurochem. 72:1466-1471. Scharnagl, H. , Tisljar, U . , Winkler, K. , Huttinger, M . , Nauck, M.A . , Gross, W., Wieland, H. , Ohm, T.G., Marz, W. 1999. The betaA4 amyloid peptide complexes to and enhances the uptake of beta-very low density lipoproteins by the low density lipoprotein receptor- related protein and heparan sulfate proteoglycans pathway. Lab Invest. 79:1271-1286. Scheer, H . W. and Lavoie, P. A . 1991. Mechanism of aminopyridine-induced release of [3H]dopamine from rat brain synaptosomes. Gen. Pharmacol. 22:169-172. Schenk, D., Barbour, R., Dunn, W., Gordon, G., Grajeda, H. , Guido, T., Hu, K. , Huang, J., Johnson-Wood, K. , Khan, K. , Kholodenko, D., Lee, M . , Liao, Z., Lieberburg, I., Motter, R., Mutter, L. , Soriano, F., Shopp, G., Vasquez, N . , Vandevert, C , Walker, S., Wogulis, M . , Yednock, T., Games, D., Seubert, P. 1999. Immunization with amyloid- beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature. 400:173- 177. Scheuner, D., Eckman, C., Jensen, M . , Song, X . , Citron, M . , Suzuki, N . , Bird, T.D., Hardy, J., Hutton, M . , Kukull, W., Larson, E., Levy-Lahad, E., Vitanen, M . , Peskind, E., Poorkaj, P., Schellenberg, G., Tanzi, R., Wasco, W., Lannfelt, L. , Selkoe, D., Younkin, S. 1996. Secreted amyloid beta-protein similar to that in the senile plaques of Alzheimer's disease is increased in vivo by the presenilin 1 and 2 and APP mutations linked to familial Alzheimer's disease. Nat Med! 2:864-870. Schilling, T. and Eder, C. 2003. Effects of kinase inhibitors on TGF-beta induced upregulation of Kv l .3 K + channels in brain macrophages. Pflugers Arch. 447:312-315. Schilling, T., Stock, C , Schwab, A . , Eder, C. 2004. Functional importance of Ca - activated K+ channels for lysophosphatidic acid-induced microglial migration. Eur. J. Neurosci. 19:1469-1474. Schlichter, L.C. , Sakellaropoulos, G., Ballyk, B. , Pennefather, P.S., Phipps, D.J. 1996. Properties of K+ and CI- channels and their involvement in the proliferation of rat microglial cells. Glia 17:225-236. Schmechel, D.E., Goldgaber, D., Burkhart, D.S., Gilbert, J.R., Gajdusek, D.C., Roses, A .D . 1988. Cellular localization of messenger R N A encoding amyloid-beta-protein in normal tissue and in Alzheimer disease. Alzheimer Dis Assoc Disord. 2:96-111. Schmechel, D.E., Saunders, A . M . , Strittmatter, W.J., Crain, B.J., Hulette, C M . , Joo, S.H., Pericak-Vance, M.A . , Goldgaber, D., Roses, A . D . 1993. Increased amyloid P- peptide deposition in cerebral cortex as a consequence of apolipoprotein E genotype in late-onset Alzheimer disease. PNAS 90:9649-9653. Schubert, W., Prior, R., Weidemann, A . , Dircksen, H. , Multhaup, G., Masters, C.L., Beyreuther, K . 1991. Localization of Alzheimer beta A4 amyloid precursor protein at central and peripheral synaptic sites. Brain Res. 563:184-194. Schumann, M . A . and Gardner, P. 1989. Modulation of membrane K + conductance in T- lymphocytes by substance P via a GTP-binding protein. J. Membr. Biol. 111:133-139. Scott, S.A., Johnson, S.A., Zarow, C , Perlmutter, L.S. 1993. Inability to detect beta- amyloid protein precursor mRNA in Alzheimer plaque-associated microglia. Exp Neurol. 121:113-118. Selkoe, D.J. 2000. Toward a comprehensive theory for Alzheimer's disease. Hypothesis: Alzheimer's disease is caused by the cerebral accumulation and cytotoxicity of amyloid beta-protein. Ann. N . Y . Acad. Sci. 924:17-25. Shaffer, L . M . , Dority, M.D. , Gupta-Bansal, R., Fredrickson, R.C., Younkin, S.G., Brunden, K.R. 1995. Amyloid beta protein (Ap) removal by neuroglial cells. Neurobiol. Aging 16: 737-745. Sheldon, C., Cheng, Y . M . , Church, J. 2004a. Concurrent measurements of the free cyctosolic concentrations of H* and N a + ions with fluorescent indicators. Pflugers Arch. 449: 307-318. Sheldon, C , Diarra, A. , Cheng, Y . M . , Church, J. 2004b. Sodium influx pathways during and after anoxia in rat hippocampal neurons. J Neurosci. 24:11057-11069. Sheng, J.G., Ito, K. , Skinner, R.D., Mrak, R.E., Rovnaghi, C.R., Van Eldik, L.J. , Griffin, W.S. 1996. In vivo and in vitro evidence supporting a role for the inflammatory cytokine interleukin-1 as a driving force in Alzheimer pathogenesis. Neurobiol Aging. 17:761-766. Sheng, W.S., Hu, S., Kravitz, F.H., Peterson, P.K., Chao, C C . 1995. Tumor necrosis factor alpha upregulates human microglial cell production of interleukin-10 in vitro. Clin Diagn Lab Immunol. 2:604-608. Sherrington, R., Rogaev, E.I., Liang, Y . , Rogaeva, E.A., Levesque, G., Dceda, M . , Chi, H. , Lin, C , L i , G., Holman, K. , et al. 1995. Cloning of a gene bearing missense mutations in early-onset familial Alzheimer's disease. Nature 375:754-760 Shibata, N . , Ohnuma, T., Takahashi, T., Baba, H. , Ishizuka, T., Ohtsuka, M . , Ueki, A . , Nagao, M . , Arai, H . 2002. Effect of IL-6 polymorphism on risk of Alzheimer disease: genotype-phenotype association study in Japanese cases. A m J Med Genet. 114:436-439. Shigematsu, K. , McGeer, P.L., McGeer, E.G. 1992. Localization of amyloid precursor protein in selective postsynaptic densities of rat cortical neurons. Brain Res. 592:353- 357. Silei, V . , Fabrizi, C , Venturini, G., Salmona, M . , Bugiani, O., Tagliavini, F, Lauro. G . M . 1999. Activation of microglial cells by PrP and beta-amyloid fragments raises intracellular calcium through L-type voltage sensitive calcium channels. Brain Res. 818:168-70. Simons, M . , Keller, P., De Strooper, B. , Beyreuther, K. , Dotti, C.G., Simons, K . 1998. Cholesterol depletion inhibits the generation of beta-amyloid in hippocampal neurons. PNAS 95:6460-6464. Snyder, S.W., Ladror, U.S., Wade, W.S., Wang, G.T., Barrett, L.W., Matayoshi, E.D., Huffaker, H.J., Krafft, G.A., Holzman, T.F. 1994. Amyloid-beta aggregation: selective inhibition of aggregation in mixtures of amyloid with different chain lengths. Biophys J. 67:1216-28. Sondag, C M . , Combs, C K . 2004. Amyloid precursor protein mediates proinflammatory activation of monocytic lineage cells. J Biol Chem. 279:14456-14463. Song, X . , Tanaka, S., Cox, D., Lee, S .C 2004. Fcgamma receptor signaling in primary human microglia: differential roles of PI-3K and Ras/ERK M A P K pathways in phagocytosis and chemokine induction. J Leukoc Biol. 75:1147-1155. Soni, N . and Kam, P. 1982. 4-aminopyridine-a review. Anaesth. Intens. Care 10:120-126. St George-Hyslop, P.H., Tanzi, R.E., Polinsky, R.J., Haines, J.L., Nee, L. , Watkins, P . C , Myers, R.H., Feldman, R.G., Pollen, D., Drachman, D. et al (1987) The genetic defect causing familial Alzheimer's disease maps on chromosome 21. Science 235:885-890 Stone, T. W. 1993. Neuropharmacology of quinolinic and kynurenic acids. Pharmacol. Rev. 45, 309-379. Strauss, S., Bauer, J., Ganter, U . , Jonas, U . , Berger, M . and Volk, B. , 1992. Detection of interleukin-6 and a2-macroglobulin immunoreactivity in cortex and hippocampus of Alzheimer's disease patients. Lab. Invest. 66: 223-230. Streit, W.J., Hurley, S.D., McGraw, T.S., Semple-Rowland, S.L. 2000. Comparative evaluation of cytokine profiles and reactive gliosis supports a critical role for interleukin- 6 in neuron-glia signaling during regeneration. J Neurosci Res. 61:10-20. Suzuki, N . , Cheung, T.T, Cai X D , Odaka A , Otvos L Jr, Eckman C, Golde TE, Younkin SG (1994) A n increased percentage of long amyloid beta protein secreted by familial amyloid beta protein precursor (beta APP717) mutants. Science 264:1336-1340. Suzumura, A . , Sawada, M . , Itoh, Y . , Marunouchi, T. 1994. Interleukin-4 induces proliferation and activation of microglia but suppresses their induction of class II major histocompatibility complex antigen expression. J. Neuroimmunol. 53:209-218. Takahashi, A . , Camacho, P., Lechleiter, J.D., Herman, B. Measurement of intracellular calcium. Physiol. Rev. 79(4): 1089-1125, 1999. Takahashi, R.H., Milner, T.A., L i , F., Nam, E.E., Edgar, M.A . , Yamaguchi, H. , Beal, M.F. , Xu , H. , Greengard, P., Gouras, G.K. 2002. Intraneuronal Alzheimer Abeta42 accumulates in multivesicular bodies and is associated with synaptic pathology. A m J Pathol. 161:1869-79. Tarkowski, E., Blennow, K. , Wallin, A . , Tarkowski, A . 1999. Intracerebral production of TNF-a a local neuroprotective agent, in Alzheimer's disease and vascular dementia. J Clin Immunol 19:223-230. Tan, J., Town, T., Paris, D., Mori, T., Suo, Z., Crawford, F., Mattson, M.P., Flavell, R.A. , Mullan, M 1999. Microglial activation resulting from CD40-CD40L interaction after beta-amyloid stimulation. Science-. 286:2352-5. Tapia, R. and Sitges, M . 1982. Effect of 4-aminopyridine on transmitter release in synaptosomes. Brain Res. 250: 291-299. Tapia, R., Sitges, M . and Morales, E.1985. Mechanism of the calcium-dependent stimulation of transmitter release by 4-aminopyridine in synaptosomes. Brain Res. 361: 373-382. Tehranian, R., Hasanvan, H. , Iverfeldt, K. , Post, C. and Schultzberg, M . 2001. Early induction of interleukin-6 mRNA in the hippocampus and cortex of APPsw transgenic mice Tg2576. Neurosci. Lett. 301:54-58. Terai K , Matsuo A , McGeer PL. Enhancement of immunoreactivity for N F - K B in the hippocampal formation and cerebral cortex of Alzheimer's disease. Neurosci Lett. 1996; 735: 159-168. Terry, R.D., Masliah, E., Salmon, D.P., Butters, N . , DeTeresa, R., Hi l l , R., Hansen, L .A. , Katzman, R. 1991. Physical basis of cognitive alterations in Alzheimer's disease: synapse loss is the major correlate of cognitive impairment. Ann. Neurol. 30:572-580. Thai, D. R., Rub, U . , Schultz, C , Sassin, I., Ghebremedhin, E., Del Tredici, K. , Braak, E., Braak, H . 2000. J. Neuropathol. Exp. Neurol. 59, 733-748. Thery, C. and Mallat, M . 1993. Influence of interleukin-1 and tumor necrosis factor alpha on the growth of microglial cells in primary cultures of mouse cerebral cortex: involvement of colony-stimulating factor 1. Neurosci Lett. 150:195-199. Thomas, T., Nadackal, G. T., Thomas, K . 2001. Aspirin and non-steroidal anti- inflammatory drugs inhibit amyloid-p* aggregation. Neuropharmacol. Neurotoxicol. 12:3263-3267. Tibbs, G. R , Dolly, J. O., Nicholls, D. G. 1989. Dendrotoxin, 4-aminopyridine, and b- bungarotoxin act at common loci but by two distinct mechanisms to induce C a 2 + - dependent release of glutamate from guinea-pig cerebrocortical synaptosomes. J. Neurochem. 52: 201-206. Tiffany HL, Lavigne M C , Cui Y H , Wang J M , Leto TL, Gao JL, Murphy P M . 2001. Amyloid-beta induces chemotaxis and oxidant stress by acting at formylpeptide receptor 2, a G protein-coupled receptor expressed in phagocytes and brain. J Biol Chem. 276:23645-23652. Tikka, T .M. , Koistinaho, J.E. 2001. Minocycline provides neuroprotection against N - mefhyl-D-aspartate neurotoxicity by inhibiting microglia. J Immunol. 166:7527-7533. Tsirka, S.E. 2002. Tissue plasminogen activator as a modulator of neuronal survival and function. Biochem Soc Trans. 30:222-225. Ueda, K. , Shinohara, S., Yagami, T., Asakura, K. , Kawasaki, K . 1997. Amyloid beta protein potentiates C a 2 + influx through L-type voltage-sensitive Ca2+ channels: a possible involvement of free radicals. J Neurochem. 68:265-271. Ujiie, M . , Dickstein, D.L, Carlow, D.A., Jefferies, W.A. 2003. Blood-brain barrier permeability precedes senile plaque formation in an Alzheimer disease model. Microcirculation. 10:463-70. Vancea, J.E., Campenotb, R.B., Vancec, D.E. 2000. The synthesis and transport of lipids for axonal growth and nerve regeneration. Biochim. Biophys. Acta 1486: 84-96. Vandenabeele, P. and Fiers, W. 1991. Is amyloidogenesis during Alzheimer's disease due to an IL-l/IL-6 mediated "acute phase" response in the brain?. Immunol Today 12: 217- 219. Vanden Berghe, W., Plaisance, S„ Boone, E., De Bosscher, K. , Schmitz, M.L . , Fiers, W., Haegeman, G. 1998. p38 and extracellular signal-regulated kinase mitogen-activated protein kinase pathways are required for nuclear factor-kappaB p65 transactivation mediated by tumor necrosis factor. J. Biol . Chem. 273:3285-3290. van Gool, W,A., Weinstein, H.C., Scheltens, P.K., Walstra, G.J. 2001. Effect of hydroxychloroquine on progression of dementia in early Alzheimer's disease: an 18- month randomised, double-blind, placebo-controlled study. Lancet. 358:455-460. Vassar, R., Bennett, B.D., Babu-Khan, S., Kahn, S., Mendiaz, E.A., Denis, P., Teplow, D.B., Ross, S., Amarante, P., Loeloff, R., Luo, Y . , Fisher, S., Fuller, J., Edenson, S., Lile, J., Jarosinski, M . A . , Biere, A . L . , Curran, E., Burgess, T., Louis, J.C., Collins, F., Treanor, J., Rogers, G., Citron, M . 1999. Beta-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease B A C E . Science. 1999 286:735- 741. Veerhuis R, Janssen I, De Groot CJ, Van Muiswinkel FL, Hack CE, Eikelenboom P. 1999. Cytokines associated with amyloid plaques in Alzheimer's disease brain stimulate human glial and neuronal cell cultures to secrete early complement proteins, but not C l - inhibitor. Exp. Neurol. 160:289-299. Veerhuis, R., Van Breemen, M.J. , Hoozemans, J.J.M., Morbin, M . , Ouladhadj, J., Tagliavini, F., Eikelenboom, P. 2003. Amyloid (3 plaque associated proteins C l q and SAP enhance the APi_42 peptide-induced cytokine secretion by adult human microglia in vitro. Acta Neuropathol. 105, 135-144. Vegeto, E., Bonincontro, C , Pollio, G., Sala, A. , Viappiani, S., Nardi, F., Brusadelli, A . , Viviani, B. , Ciana, P., Maggi, A . 2001. Estrogen prevents the lipopolysaccharide-induced inflammatory response in microglia. J Neurosci. 21:1809-1818. Vekrellis, K. , Ye, Z., Qiu, W.Q., Walsh, D., Hartley, D., Chesneau, V . , Rosner, M.R., Selkoe, D.J. 2000. Neurons regulate extracellular levels of amyloid beta-protein via proteolysis by insulin-degrading enzyme. J. Neurosci. 20:1657-1665. Verdier, Y . , Zarandi, M . , Penke, B. 2004. Amyloid beta-peptide interactions with neuronal and glial cell plasma membrane: binding sites and implications for Alzheimer's disease. J Pept Sci. 10:229-248. Verkhratsky, A . , Orkand, R.K., Kettenmann, H . 1998. Glial calcium: homeostasis and signaling function. Physiol Rev. 78:99-141. Viel , J.J., McManus, D.Q., Smith, S.S., Brewer, G.J. 2001. Age- and concentration- dependent neuroprotection and toxicity by TNF in cortical neurons from beta-amyloid. J. Neurosci. Res 64:454—465. Visentin, S., Renzi, M . , Levi, G. 2001. Altered outward-rectifying K(+) current reveals microglial activation induced by HIV-1 Tat protein. Glia. 33:181-190. von Strauss, E., Viitanen, M . , De Ronchi, D., Winblad, B. , Fratiglioni, L . 1999. Aging and the occurrence of dementia: findings from a population-based cohort with a large sample of nonagenarians. Arch Neurol. 56:587-592. Walker, D.G., Kim, S.U., McGeer, P.L. 1995. Complement and cytokine gene expression in cultured microglia derived from post-mortem human brains. J. Neurosci. Res. 40, 478- 493. Walker, D.G., Kim, S.U., McGeer, P.L. 1995. Complement and cytokine gene expression in cultured microglial derived from postmortem human brains. J. Neurosci. Res. 40:478- 493. Walker, D.G., Lue, L.F. and Beach, T.G. 2001. Gene expression profiling of amyloid beta peptide-stimulated human post-mortem brain microglia. Neurobiol. Aging. 22, 957- 966. Walsh, D .M. , Hartley, D .M. , Kusumoto, Y . , Fezoui, Y . , Condron, M . M . , Lomakin, A . , Benedek, G.B., Selkoe, D.J., Teplow, D.B. 1999. Amyloid beta-protein fibrillogenesis. Structure and biological activity of protofibrillar intermediates J Biol Chem. 274:25945- 52. Walsh, D .M. , Tseng, B.P., Rydel, R.E., Podlisny, M.B. , Selkoe, D.J. 2000. The oligomerization of amyloid beta-protein begins intracellularly in cells derived from human brain. Biochemistry. 39:10831-10839. Walz, W., Ilschner, S., Ohlemeyer, C , Banati, R., Kettenmann, H . 1993. Extracellular ATP activates a cation conductance and a K+ conductance in cultured microglial cells from mouse brain. J Neurosci. 13:4403-4411. Webster S., Lue, L.F., Brachova, L. , Tenner, A.J . , McGeer, P.L., Terai, K. , Walker, D.G., Bradt, B. , Cooper, N.R., Rogers J. 1997. Molecular and cellular characterization of the membrane attack complex, C5b-9, in Alzheimer's disease. Neurobiol. Aging 18:415—421. Webster, S., O'Barr, S., Rogers, J. 1994. Enhanced aggregation and beta structure of amyloid beta peptide after coincubation with C l q . J. Neurosci. Res. 39: 448—456. Weggen, S., Eriksen, J. L. , Sagi, S. A. , Pietrzik, C. U . , Ozols, V . , Fauq, A. , Golde, T. E., Koo, E. H . 2003. Evidence that nonsteroidal anti-inflammatory drugs decrease A(542 production by direct modulation of y-secretase activity. J. Biol. Chem. 278: 31831-31837. Weiss, J.H., Pike, C.J., Cotman, C.W. 1994. C a 2 + channel blockers attenuate beta- amyloid peptide toxicity to cortical neurons in culture. J Neurochem. 62:372-375. Weldon, D.T., Rogers, S.D., Ghilardi, J.R., Finke, M.P., Cleary, J.P., O'Hare, E., Esler, W.P., Maggio, J.E., Mantyh, P.W. 1998. Fibrillar beta-amyloid induces microglial phagocytosis, expression of inducible nitric oxide synthase, and loss of a select population of neurons in the rat CNS in vivo. J. Neurosci. 18:2161-2173. Wesseling, H. , Agoston, S., Van Dam, G.B., Pasma, J., DeWit, D.J., Havinga, H . 1984. Effects of 4-aminopyridine in elderly patients with Alzheimer's disease. N Engl J Med. 310:988-9. Wiseman, E.J., Jarvik, L.F. 1991. Potassium channel blockers: could they work in Alzheimer disease? Alzheimer Dis Assoc Disord. 5:25-30. Wolfe, M.S., Xia , W., Ostaszewski, B.L. , Diehl, T.S., Kimberly, W.T., Selkoe, D.J. 1999. Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and gamma-secretase activity. Nature 398:513-517 Wolozin, B. , Kellman, W., Ruosseau, P., Celesia, G.G., Siegel, G. 2000. Decreased prevalence of Alzheimer disease associated with 3-hydroxy-3-methyglutaryl coenzyme A reductase inhibitors. Arch Neurol. 57:1439-1443. Wyss-Coray, T., Lin, C , Yan, F., Yu , G.Q., Rohde, M . , McConlogue, L. , Masliah, E., Mucke, L. 2001. TGF-betal promotes microglial amyloid-beta clearance and reduces plaque burden in transgenic mice. Nat. Med. 7:612-618. Wyss-Coray, T., and Mucke, L . 2002. Inflammation in neurodegenerative disease—a double-edged sword. Neuron. 35:419-432. Xia , M.Q. and Hyman, B.T. 1999. Chemokines/chemokine receptors in the central nervous system and Alzheimer's disease. J. Neurovirol. 5:32—41. Xia , M . , Qin, S., McNamara, M . , Mackay, C. and Hyman, B.T. 1997. Interleukin-8 receptor B immunoreactivity in brain and neuritic plaques of Alzheimer's Disease, Amer. J. Path. 150, 1267-1274. Xia, M.Q. Qin, S.X. Wu, L.J . Mackay C.R. and Hyman, B.T. 1998. Immunohistochemical study of the beta-chemokine receptors CCR3 and CCR5 and their ligands in normal and Alzheimer's disease brains. Am. J. Pathol. 153:31-37. Xie, Z., Wei, M . , Morgan, T.E., Fabrizio, P., Han, D., Finch, C.E., Longo, V . D . 2002. Peroxynitrite mediates neurotoxicity of amyloid beta peptide 1-42- and lipopolysaccharide-activated microglia. J Neurosci. 22:3484-92. Yamazaki, T., Koo, E. H. , and Selkoe, D. J. 1997. Cell surface amyloid beta-protein precursor colocalizes with beta 1 integrins at substrate contact sites in neural cells. J. Neurosci. 17:1004-1010 Yan, S.D., Chen, X . , Fu, J., Chen, M . , Zhu, H. , Roher, A . , Slattery, T., Zhao, L. , Nagashima, M . , Morser, J., Migheli, A . , Nawroth, P., Stern, D., Schmidt, A . M . 1996. R A G E and amyloid-beta peptide neurotoxicity in Alzheimer's disease. Nature. 382:685- 691. Yao, J., Harvath, L. , Gilbert, D.L., Colton, C A . 1990. Chemotaxis by a CNS macrophage, the microglia. J Neurosci Res. 27:36-42. Yasojima, K. , Schwab, C , McGeer, E.G., McGeer, P L . 1999. Distribution of cyclooxygenase-1 and cyclooxygenase-2 mRNAs and proteins in human brain and peripheral organs. Brain Res. 830:226-236. Yasojima, K. , Schwab, C , McGeer, E.G., McGeer, P L . 2000. Human neurons generate C-reactive protein and amyloid P: upregulation in Alzheimer's disease. Brain Res. 887, 80-89. Yates, S.L., Burgess, L .H. , Kocsis-Angle, J., Antal, J .M., Dority, M.D. , Embury, P.B., Piotrkowski, A . M . , Brunden, K.R. 2000. Amyloid beta and amylin fibrils induce increases in proinflammatory cytokine and chemokine production by THP-1 cells and murine microglia. J Neurochem. 74:1017-1025. Ye, C , Ho-Pao, C.L., Kanazirska, M . , Quinn, S., Rogers, K. , Seidman, C.E., Seidman, J.G., Brown, E .M. , Vassilev, P .M. 1997. Amyloid-beta proteins activate Ca2+-permeable channels through calcium-sensing receptors. J Neurosci Res. 47:547-54. Yermakova, A . V . , O'Banion, M . K . 2001. Downregulation of neuronal cyclooxygenase-2 expression in end stage Alzheimer's disease. Neurobiol Aging 22:823-36. Young, J. D.-E., Unkeless, J.C., Kaback, H.R., Cohn, Z.A.I983a. Macrophage membrane potential changes associated with gamma 2b/gamma 1 Fc receptor-ligand binding. PNAS U.S.A. 80:1357-1361. Young, J. D.-E., Unkeless, J.C., Young, T .M. , Mauro, A . , Cohn, Z.A. 1983b. Role for mouse macrophage IgG Fc receptor as ligand-dependent ion channel. Nature 306:186- 189. Zhao, M.L . , Liu, J.S., He, D., Dickson, D.W., Lee, S.C. 1998. Inducible nitric oxide synthase expression is selectively induced in astrocytes isolated from adult human brain. Brain Res. 813:402-405. Zemlan, F.P., Thienhaus, O.J., Bosmann, H.B. 1989. Superoxide dismutase activity in Alzheimer's disease: possible mechanism for paired helical filament formation. Brain Res. 476:160-162. Zujovic, V . , Benavides, J., Vige, X . , Carter, C , Taupin, V . 2000. Fractalkine modulates TNF-alpha secretion and neurotoxicity induced by microglial activation. Glia. 29:305- 315.

Cite

Citation Scheme:

    

Usage Statistics

Country Views Downloads
Japan 3 0
France 2 0
United States 2 2
China 2 25
City Views Downloads
Tokyo 3 0
Beijing 2 0
Unknown 2 4
Redmond 1 0
Ashburn 1 0

{[{ mDataHeader[type] }]} {[{ month[type] }]} {[{ tData[type] }]}

Share

Share to:

Comment

Related Items