Abstract i List of contents iii List of figures vi List of abbreviation viii
Ⅰ. Introduction 1 1.1 Microglia 1 1.1.1 The discovery and definition of microglia 1 1.1.2 Origin and development of microglia 4 1.1.3 Physiological and pathological roles of microglia 6 1.1.4 Microglia in regulation of energy homeostasis and development of meta-bolic syndromes 8 1.2 Inflammasome 10 1.2.1 Principle of inflammasome activation 10 1.2.2 Role of microglial inflammasome in CNS 15 1.3 Casein Kinase 2 (CK2) 17 1.4 Translocator protein (TSPO) 19 Ⅱ. A translocator protein 18 kDa ligand, Ro5-4864, inhibits ATP-induced NLRP3 inflam-masome activation 21 2.1 Introduction 21 2.2 Materials and methods 23 2.2.1 Reagents and antibodies 23 2.2.2 Cell culture and preparation 23 2.2.3 Induction of NLRP3 inflammasome activation 24 2.2.4 Enzyme-linked immunosorbent assay (ELISA) 24 2.2.5 Western blotting and ASC oligomerization analyes 24 2.2.6 Immunocytochemistry analysis 25 2.2.7 Measurement of mitochondria membrane potential and mitochondrial superoxide production 26 2.2.8 Statistics 26 2.3 Results 27 2.3.1 Ro5-4864 strongly inhibits NLRP3 inflammasome activation triggered by ATP in LPS-primed THP-1 and BMDM cells Reagents and antibodies 27 2.3.2 Ro5-4864 potently reduces NLRP3 inflammasome assembly triggered by LPS/ATP 30 2.3.3 Ro5-4864 inhibits mitochondrial association of NLRP3 32 2.3.4 Ro5-4864 attenuates ATP-induced perturbation of mitochondrial function in LPS-primed THP-1 cells 34 2.4 Discussion 37 Ⅲ. Microglial CK2 is critical for metabolic homeostasis and obesity prevention by ASC phosphorylation and inflammasome-dependent IL-18 processing 40 3.1 Introduction 40 3.2 Materials and methods 45 3.2.1 Reagents and resource table 45 3.2.2 Mice 48 3.2.3 Metabolic measurements 48 3.2.4 Fat and Lean Mass measurement 49 3.2.5 Glucose tolerance test and insulin tolerance test 49 3.2.6 Primary microglia and BMDM cell cultures 49 3.2.7 Cell culture 50 3.2.8 Isolation of CD11b-positve cells 50 3.2.9 Inflammasome activation 51 3.2.10 Cerebrospinal fluid (CSF) and serum collection 51 3.2.11 Cytokine measurements 52 3.2.12 Immunoblotting 52 3.2.13 Phos-tag gel 53 3.2.14 Genomic DNA isolation and genotyping 53 3.2.15 Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) 53 3.2.16 Site-directed mutagenesis 54 3.2.17 Live imaging for TMRE 54 3.2.18 Overexpression of hASC-GFP 54 3.2.19 Immunocytochemistry 55 3.2.20 Ex vivo brain slice experiment and immunohistochemistry (IHC) 55 3.2.21 Identification of ASC phosphorylation by LC/MS 56 3.2.22 Purification of GST-hASC 57 3.2.23 CK2 in vitro kinase assay 58 3.2.24 Statistical analysis 58 3.3 Results 59 3.3.1 Microglia-selective deletion of Csnk2b leads to obesity 59 3.3.2 Pharmacological and genetic inaction of CK2 reduces IL-18 and IL-1β matura-tion and secretion by inhibiting NLRP3 inflammasome activation 67 3.3.3 Inactivation of CK2 inhibits signal 2-triggered activation step, not signal 1-induced priming of NLRP3 inflammasome 75 3.3.4 Inactivation of CK2 does not block mitochondrial dysfunctions or α-tubulin acetylation 78 3.3.5 CK2 is required for ASC oligomerization 80 3.3.6 CK2 phosphorylates ASC at T154 82 3.4 Discussion 90 Ⅳ. Conclusion 94 V. Reference 95 Ⅵ. Abstract in Korean 106