From DEPARTMENT OF NEUROBIOLOGY, CARE SCIENCE AND SOCIETY Karolinska Institutet, Stockholm, Sweden INFLAMMATION AS A THERAPEUTIC TARGET FOR ALZHEIMER’S DISEASE ERIK HJORTH Stockholm 2010 All previously published papers were reproduced with permission from the publisher. Published by Karolinska Institutet. Printed by [name of printer] The author may be contacted on [email protected] © Erik Hjorth, 2010 ISBN 978-91-7409-883-9 ABSTRACT Alzheimer‟s disease (AD) is a progressive neurodegenerative disorder which is characterised by impairment of memory and learning. The impairment is caused by neuronal death which originates in the parts of the brain that execute memory functions: the entorhinal cortex and hippocampus. The neuronal death is believed to be caused by the amyloid- (A ) peptide which is prone to oligomerisation and aggregation into insoluble amyloid plaques (AP). The levels of soluble A and the number of AP:s are increased in the AD brain which is attributed to increased production and impaired clearance of A . Another hallmark of AD, after neuronal death and the increased presence of A , is inflammation in the form of activated microglia and increased levels of inflammatory proteins in the brain. Inflammation in the CNS has been shown to increase the production of A and to impair, and even kill, neurons. On the other hand, inflammation has been shown to increase the removal of pathogens, such as A , from the brain by increasing the phagocytic acticity of microglia. Inflammation is also associated with an increased secretion of neurotrophic factors that can protect neurons. Somehow this clearance of A is impaired in AD and the levels of neurotrophic factors are decreased. The work on this thesis has been focused on the inflammatory component of AD and how it can be modulated into performing activities that are beneficial for neurons without evoking the harmful activities of inflammation. For this purpose a human microglial cell line, CHME3, was characterized with regard to interaction with A and inflammatory responses. The CHME3 microglial cell line was used as a model of inflammation in the human brain. In this model, we tested the ability of the neuropeptide -MSH to inhibit inflammation induced by A 1-40 and cytokines, and found that -MSH, on the contrary, increased secretion of the inflammatory cytokine interleukin (IL)-6. -MSH was also found to protect human neuronal cells against necrotic stress. The possibility of stimulating microglia into phagocytosis of A 1-42 and the concurrent responses was also investigated. We found that the omega-3 fatty acid DHA and the adjuvant protollin had a stimulatory effect on phagocytosis of A 1-42. DHA also had an inhibitory effect on the secretion of several cytokines. Microglia that were phagocytic of A 1-42 displayed increased expression of several inflammatory 1 markers compared to non-phagocytic cells. Thus, inducible nitric oxide synthase (iNOS) was expressed to a larger extent on phagocytic cells. Treament with DHA abolished this imbalance in iNOS expression, indicating a less harmful phenotype of phagocytic cells upon treatment with DHA. Furthermore, inflammation in the form of IFN stimulated phagocytosis of A 1-42 while decreasing the secretion of the neurotrophin brain-derived neurotrophic factor and inducing microglial cell death. A 1- 42 was also found to decrease the secretion of BDNF. The effect of omega-3 supplement on the levels of A 1-42, tau and inflammatory markers in the cerebrospinal fluid and plasma of AD-patients was investigated in an intervention study. There were no detectable differences in any of the markers after 6 months of treatment with omega- 3 compared with placebo control. However, at base line, a significant positive correlation between the soluble interleukin (IL)-1 receptor type II was observed. The major finding in this thesis is the reduction in BDNF induced by A 1-42, and the AD-related cytokine IFN , suggesting a new pathogenic mechanism in AD. Other important findings are the beneficial effect of DHA on microglial activities, which support the use of omega-3 supplements for treatment of AD. However, the results from the intervention study did not provide additional support for the use of omega-3, but suggest the importance of early intervention. -MSH was found to promote neuronal survival suggesting a potential role for this peptide and its receptors for the treatment of neurodegenerative disorders. 2 LIST OF PUBLICATIONS I. Lindberg C, Hjorth E, Post C, Winblad B, Schultzberg M Cytokine production by human microglia: effects of -amyloid and - melanocyte stimulating hormone Neurotoxicity Research, 2005, 8(3,4), 267-276 II. Hjorth E, Frenkel D, Weiner H, Schultzberg M Effects of immunomodulatory substances on phagocytosis of A 1-42 by human microglia International Journal of Alzheimer’s disease (accepted) III. Freund-Levi Y, Hjorth E, Lindberg C, Cederholm T, Faxen-Irving G, Vedin I, Palmblad J, Wahlund L-O, Schultzberg M, Basun H, Eriksdotter Jönhagen M Effects of Omega-3 Fatty Acids on Inflammatory Markers in Cerebrospinal Fluid and Plasma in Alzheimer‟s Disease: The OmegAD Study Dement Geriatr Cogn Disord 2009;27:481–490 IV. Hjorth E, Vedin I, Palmblad J, Cederholm T, Freund-Levi Y, Faxen-Irving G, Wahlund L-O, Basun H, Eriksdotter Jönhagen M and Schultzberg M Effects of omega-3 fatty acids on microglial phagocytosis and inflammatory phenotype Manuscript 3 CONTENTS 1 Introduction ................................................................................................................. 7 1.1 Alzheimer‟s disease .......................................................................................... 7 1.2 The immune system and inflammation ............................................................ 9 1.2.1 Molecular mediators of the immune response .................................. 13 1.2.2 Intracellular signalling and gene activation in inflammation ............ 14 1.3 Inflammation in the central nervous system .................................................. 15 1.3.1 Damage and impairment of neurons caused by inflammation.......... 16 1.3.2 Cellular mediators of inflammation in the CNS ................................ 17 1.4 Microglial activities in inflammation ............................................................. 18 1.4.1 Secretory products .............................................................................. 18 1.4.2 Phagocytosis and degradation of debris and pathogens .................... 18 1.4.3 Inflammation and AD ........................................................................ 20 1.5 Modulation of the inflammatory response into a therapy for AD ................. 21 2 Thesis aims ............................................................................................................... 22 2.1 Specific aims ................................................................................................... 22 3 Methodology ............................................................................................................. 23 3.1 General overview ............................................................................................ 23 3.1 Cellular models – papers I, II, IV and preliminary results ............................ 23 3.1.1 A model of human microglia: The human CHME3 microglial cell line ............................................................................................................. 23 3.1.2 A model for neurodegeneration on human neuronal cells ................ 24 3.1.3 Immunochemistry ............................................................................... 24 3.2 Effects of intervention with EPAX on inflammatory markers in AD-patients – a clinical randomized trial ........................................................................... 29 3.2.1 The study population .......................................................................... 29 3.2.2 Study design ....................................................................................... 30 3.3 Introduction to the immunomodulatory substances USED ........................... 30 3.3.1 Protollin .............................................................................................. 30 IL-1 ................................................................................................... 31 IFN .................................................................................................... 31 3.3.4 DHA and EPA .................................................................................... 32 3.3.5 EPAX and placebo ............................................................................. 32 3.3.6 Melanocyte stimulating hormone ( MSH) ................................ 32 4 Results and discussion .............................................................................................. 35 4.1 Characterization of human CHME3 microglia with regard to interactions with A 1-42 and inflammation......................................................................... 36 4.1.1 Secretion of IL-6 from human CHME3 microglial cells .................. 36 4.1.2 Secretion of BDNF from human CHME3 microglial cells .............. 37 4.1.3 Cell proliferation and cell death ......................................................... 39 4.1.4 Phagocytosis ....................................................................................... 40 4.1.5 Microglial phenotype and relation to phagocytosis .......................... 41 4.2 Immunomodulation of responses related to neuroprotection