진병관 박근우 2008-02 6400 https://aurora.ajou.ac.kr/handle/2018.oak/17153 학위논문(박사)----아주대학교 일반대학원 :신경과학기술과정,2008. 2 Microglia는 중추신경계에 존재하는 면역세포로 뇌손상시에 활성화된다. Microglia의 활성화는 감염에 대항하는 방어기전이지만 활성화된 microglia로부터 생성되는 염증 매개 물질들이 뇌의 손상을 가중시켜 뇌질환의 발병 및 진전을 악화시킬 수 있으므로 이러한 염증반응의 정도 및 기간의 조절은 매우 중요하게 여겨진다. 대표적인 항염증성 사이토 카인인 IL-13과 IL-4는 염증 매게 물질의 생성을 억제하는 것으로 보고되어 왔다. 이러한 IL-13과 IL-4의 염증 메게 물질의 생성 억제 효과 이외의 연구 결과도 보고 되었는데 최근 보고에 따르면 IL-13과 IL-4는 활성화된 microglia의 사멸에 관여하는 것으로 밝혀졌다. 본 연구의 목적은 동물 내 해마와 대퇴 피질에서의 IL-13과 IL-4의 역할을 확인하고 신경세포에서의 NADPH oxidase에 의한 활성산소의 생성이 세포사멸을 유도할 수 있는지 확인하는 것이다. 우선, 해마에서의 IL-13과 IL-4의 역할을 확인하기 위해 트롬빈과 amyloid beta를 이용하여 실험을 수행하였다. Alzheimer’s disease와 관련이 높은 것으로 알려진 트롬빈과 amyloid beta를 해마에 주입한 후 신경세포의 사멸 유도와 microglia의 활성화를 면역화학염색법을 이용하여 확인하였고 이후 IL-13과 IL-4발현을 확인한 결과 이들의 발현이 증가됨을 관찰하였다. 이러한 싸이토카인의 발현은 활성화된 microglia에만 나타남을 확인되었는데 이때 IL-13과 IL-4의 역할을 확인하기 위해 IL-13과 IL-4의 neutralizing antibody (NA) 를 트롬빈이나 amyloid beta과 같이 주입한 후 신경세포의 사멸을 관찰한 결과 IL-13 NA과 IL-4 NA에 의해 신경세포의 사멸이 감소함을 확인하였다. 이때 대표적 염증 매게 물질인 iNOS, TNF-α, IL-1β의 변화를 RT-PCR과 western blot으로 확인한 결과 IL-13 NA과 IL-4 NA 에 의해 감소됨을 확인하였다. 또한 NADPH oxidase의 두 구성요소인 (p47phox, p67phox)의 발현이 IL-13NA과 IL-4NA에 의해 감소됨이 관찰되었고, 이의 결과물인 활성산소의 생성 또한 감소됨이 확인되었다. 다음으로, 트롬빈에 의한 신경세포의 사멸에 NADPH oxidase 활성화를 통한 활성산소의 생성이 관여함을 확인하기 위해 세포 배양 모델에서 실험을 수행하였다. 해마 신경 세포 배양체에 트롬빈을 처리한 후 신경세포의 사멸을 관찰하였고 이때 활성산소의 생성과 NADPH oxidase의 발현이 증가함을 확인하였다. 트롬빈에 의해 유도된 활성산소의 생성과 NADPH oxidase가 신경세포의 사멸에 관여함을 확인하기 위하여 NADPH oxidase의 억제제인 apocynin을 전처리 한 후 트롬빈을 처리해 본 결과 트롬빈에 의해 유도된 활성산소의 생성이 감소함을 확인하였고 또한 apocynin에 의해 트롬빈에 의해 유도되는 신경세포의 사멸 또한 감소함을 확인함으로써 트롬빈에 의해 유도되는 NADPH oxidase생성이 활성산소의 생성을 통해 신경세포의 사멸을 유도함을 확인할 수 있었다. 마지막으로, 흰쥐의 대퇴 피질에서의 IL-4의 발현과 역할을 확인하기 위한 실험을 수행 하였다. 흰쥐의 대뇌피질에 LPS를 주입 하였을 때 내제적 IL-4의 발현 증가와 microglia의 사멸이 관찰되었고 이러한 IL-4의 발현은 활성화된 microglia에서만 확인 되었다. IL-4의 기능을 확인하기 위해 IL-4의 blocking antibody인 neutralizing antibody를 LPS 와 함께 주입하였을 때 microglia의 사멸이 감소됨을 확인하였고 또한 신경세포의 사멸이 증가됨을 확인하였다. 종합하여 보면, IL-13과 IL-4는 해마 내에서는 NADPH oxidase의 활성화를 통한 활성산소의 생성에 관여함으로써 신경세포의 사멸에 관여하고 있음을 확인하였고 반면 대뇌 피질에서는 IL-4가 microglia의 사멸에 관여함으로써 신경세포의 생존에 도움이 됨을 확인하였다. 따라서 본 논문에서는 IL-13과 IL-4의 기능이 뇌 내의 위치에 따라 유익할 수도 반대로 유해할 수도 있다는 결과를 보여주고 있다. I. INTRODUCTION = 1 1. Origin of microglia = 1 2. Characterization of microglia = 1 2.1 Morphological change = 2 2.2 Change of surface antigen = 2 3. Protective role of mciroglia = 3 3.1 Anti-inflammatory cytokines = 4 3.1.1 IL-13 = 4 3.1.2 IL-4 = 5 4. Harmful effect of microglia = 6 4.1 Reactive oxygen species = 7 4.2 Pro-inflammatory cytokines = 8 5. Microglial death as an inflammatory resolution = 8 6. Involvement of microglia activation in Alzheimer’s Disease = 9 7. Multiple pathway leading to microglial activation = 10 8. Aims of study = 12 II. MATERIAL AND MEHTODS = 13 1. Chemicals = 13 2. Stereotaxic surgery and drug injection = 13 3. Neuron-enriched hippocampal culture = 14 4. Tissue preparation and immunohistochemistry = 15 5. Double-immunofluorescence staining = 16 6. Tomato Lectin Histochemistry = 17 7. Terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick-end labeling (TUNEL) assay = 18 8. MTT reduction assay = 18 9. Real-time/ reverse transcription?polymerase chain reaction = 19 10. Western blot analysis = 20 11. Measurement of ROS generation = 22 12. Detection of protein oxidation = 23 13. Measurement of the densities of the immunoblot bands = 24 14. Measurement of IL-13/ IL-4 = 24 15. Counting of hippocampal CA1 neurons = 25 16. Statistical analysis = 25 III. RESULTS = 26 Part A. IL-13/ IL-4 regulate oxidative stress via activation of NADPH oxidase and degeneration of hippocampal neurons in thrombin-treated hippocampus in vivo = 26 1. Thrombin induces neurodegeneration in the hippocampus = 26 2. Microglial activation and detection of IL-13/ IL-4 in thrombin-injected hippocampus = 28 3. Thrombin induced IL-13/ IL-4 expression in activated microglia in vivo = 31 4. IL-13/ IL-4 contributes to thrombin-induced neuronal degeneration in vivo = 31 5. Effect of IL-13 NA/ IL-4 NA on thrombin-induced proinflammatory cytokine and iNOS expression = 33 6. Thrombin-induced NADPH oxidase-mediated production of ROS was inhibited by IL-13 NA/ IL-4 NA = 36 Part B. IL-13/ IL-4 regulate oxidative stress via activation of NADPH oxidase and degeneration of hippocampal neurons in Aβ1-42-treated hippocampus in vivo = 43 1. Aβ1-42 induces neurodegeneration in the hippocampus = 43 2. Microglial activation and detection of IL-13/ IL-4 in Aβ1-42-injected hippocampus = 45 3. IL-13/ IL-4 expression in activated microglia in vivo = 47 4. IL-13/ IL-4 expression in APP/PS1 Tg-mouse = 47 5. IL-13/ IL-4 contributes to Aβ1-42-induced neurodegeneration in vivo = 47 6. IL-13/ IL-4 mediates ROS production and oxidative damage in the hippocampus in vivo.......= 49 Part C. Oxidative stress via activation of NADPH oxidase mediates thrombin-induced neurotoxity in hippocampal neuronal culture = 55 1. Thrombin induces neurodegeneration in neuron-enriched hippocampal cultures = 55 2. Thrombin induces ROS production and NADPH oxidase expression in neuron-enriched hippocampal cultures = 57 3. Thrombin induces the NADPH oxidase-mediated production of ROS in neuron-enriched hippocampal cultures = 64 4. NADPH oxidase contributes to thrombin-induced neurodegeneration in hippocampal cultures in vitro = 65 Part D. Microglia expressing IL-4 undergo cell death and contributes to neuronal survival in rat cortex = 69 1. LPS induces microglial cell death and IL-4 expression in the rat cerebral cortex = 69 2. IL-4 expression in activated microglia in vivo = 71 3. IL-4 contributes to the degeneration of microglia in the cerebral cortex in vivo = 71 4. IL-4 blockade exacerbates neuronal cell death in the cortex in vivo = 73 IV. DICUSSION = 77 Part A. IL-13/ IL-4 regulate oxidative stress via activation of NADPH oxidase and degeneration of hippocampal neurons in thrombin-treated hippocampus in vivo = 77 Part B. IL-13/ IL-4 regulate oxidative stress via activation of NADPH oxidase and degeneration of hippocampal neurons in Aβ1-42-treated hippocampus in vivo = 81 Part C. Oxidative stress via activation of NADPH oxidase mediates thrombin-induced neurotoxity in hippocampal neuronal culture = 83 Part D. Microglia expressing IL-4 undergo cell death and contribute to neuronal survival in rat cortex = 87 V. SUMMARY AND CONCLUSION = 91 REFERENCE = 92 국문요약 = 111 eng The Graduate School, Ajou University 아주대학교 논문은 저작권에 의해 보호받습니다. Microglia 활성화와 신경세포 사멸에서의 Interleukin-13 /Interleukin-4 의 역할 Park, Keun Woo Thesis 아주대학교 일반대학원 Park, Keun Woo 일반대학원 신경과학기술과정 2008. 2 Master http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000006400 LPS Aβ1-42 Thrombin IL-13 IL-4 ROS NADPH oxidase microglia neuron hippocampus cortex Although the major role of inflammation is host defence against infection or injured tissue in the central nervous system (CNS), inflammatory mediators can also damage the surrounding tissue and aggravate injury. Thus, endogenous mechanisms for resolution of inflammation should exist to control the duration and extent of inflammation. Increasing evidence indicates that IL-13/ IL-4, well-known anti-inflammatory cytokines, has been shown to reduce the production of inflammatory mediators. In addition, recent studies have suggested that IL-13/ IL-4 control brain inflammation by inducing the death of activated microglia in vitro. The purpose of this study was to investigate the role of endogenous IL-13 and/or IL-4 in cortex and hippocampus in vivo and the possible role of neuronal NADPH oxidase mediated- reactive oxygen species (ROS) generation in thrombin-induced neuronal degeneration in vitro. In first part, in the rat hippocampus, thrombin or amyloid beta1-42 (Aβ1-42) injection resulted in a significant neurodegeneration of CA1 hippocampal layer, accompanied by the substantial microglial activation. Interestingly, immunohistochemical and biochemical evidence demonstrated that intrahippocampal injection of thrombin or Aβ1-42 led to upregulation of IL-13/ IL-4 immunoreactivity and increased level of IL-13/ IL-4. Moreover, double-label immunohistochemistry revealed that IL-13/ IL-4 immunoreactivity was detected exclusively in activated microglia. Consistent with results from A?-injected tissues, IL-13/ IL-4 immunoreactivity were also localized within activated microglia in APP/PS1 transgenic mice. In experiments designed to determine the involvement of IL-13/ IL-4 in neurotoxicity, IL-13-neutralizing antibodies (NA)/ IL-4 NA significantly increased survival of CA1 hippocmapal neurons after injection. This neuroprotective effect was accompanied by reducing nitric oxide synthase (iNOS) and several proinflammatory cytokines. Consistent with these results, IL-13 NA/ IL-4 NA inhibited activation of NADPH oxidase, evidenced by decrease in translocation of cytosolic proteins (p67phox and p47phox) to the membrane, generation of ROS and oxidative damage. In addition to these in vivo data, thrombin can also induce the production of ROS through activation of neuronal NADPH oxidase, and this contributes to oxidative damage and consequently to neurodegeneration in hippocampal neuronal cultures. Immunocytochemical and biochemical evidence demonstrated that in neuron-enriched hippocampal cultures, thrombin induces neurodegeneration in a dose-dependent manner. In parallel, ROS production was evident as assessed by analyzing DCF and hydroethidine. Real-time PCR analysis demonstrated that expression of NADPH oxidase subunits (p47phox, p67phox, and gp91phox) occurs. In addition, Western blot analysis and double-label immunocytochemistry showed an upregulation in the expression of cytosolic components (Rac 1 and p67phox), the translocation of cytosolic proteins (p67phox and p47phox) to the membrane, and the localization of gp91phox or p47phox expression in hippocampal neurons of cultures and CA1 layer. The thrombin-induced ROS production, protein oxidation and loss of cultured hippocampal neurons were partially attenuated by an NADPH oxidase inhibitor and/or by several antioxidants. Although I demonstrated that IL-13/ IL-4 have harmful effect in thrombin or Aβ1-42 injected hippocampus, LPS injection into the rat cortex led to increased levels of endogenous IL-4 expression, followed by the substantial loss of microglia. IL-4 immunoreactivity was detected exclusively within activated microglia, but not in neurons or astrocyte. In experiments designed to determine the involvement of IL-4 in activated microglia cell death, co-injection of IL-4 NA with LPS significantly increased survival of activated microglia. Consistent with these results, the expression of inducible iNOS and tumor necrosis factor (TNF-α) was sustained in activated microglia and neuronal cell death was consequently increased. Collectively, the present study demonstrates that IL-13/ IL-4 may regulate ROS generation through activation of NADPH oxidase and consequently contribute to degeneration of hippocampal neurons in vivo, although in the cortex IL-4 induce the death of activated microglia. Taken together, the results in this thesis show that IL-13/ IL-4 have both beneficial and harmful effects on neurons, depending on different region including cerebral cortex and hippocampus and neuronal oxidative stress via NADPH oxidase activation contributes to the neuronal death.