Studies on the thrombin-induced microglia activation and neurodegeneration: Comparison between proteolytic and non-proteolytic action
Thrombin에 의한 마이크로글리아의 활성화와 신경세포의 사멸
- 발행기관 Graduate School of Ajou University
- 지도교수 Jin, Byung Kwan
- 발행년도 2005
- 학위수여년월 2005. 2
- 학위명 박사
- 학과 및 전공 일반대학원 신경과학기술과정
- 본문언어 영어
초록/요약
Thrombin is a member of the serine protease family that plays multifunctional roles in wound healing and blood coagulation and can be generated from endogeneous prothrombin. Thrombin can be extravasated into the brain following the breakdown of the blood-brain barrier (BBB). Increasing evidences indicate that thrombin has a lot of physiological roles in neurons and glial cells. Previous studies reported that thrombin can act as a neurotoxin in many types of neurons including hippocampal and spinal motor neurons. In addition, thrombin also known as a microglia activator resulting in the production of pro-inflammatory cytokines. The physiological effects of thrombin are mediated by means of G-protein-coupled receptors known as protease activated receptors (PARs). The purpose of this study was to determine the mechanisms underlying thrombin-induced neuronal cell death and microglial activation. Immunocytochemical and biochemical evidence indicated that thrombin led to profound loss of mesencephalic and cortical neurons in the presence of microglia. Accompanying neurodegeneration, microglial activation was obvious in vivo and in vitro, evidenced by OX-6-immunohistochemistry and expression of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and by increasing production of TNF-α and nitric oxide (NO). In mesencephalic neurons treated with conditioned media taken from thrombin-activated microglia, the number of dopaminergic neurons was significantly attenuated. However, the agonist peptides of thrombin receptors (PAR-1, -3 and -4) such as SFLLRN, TFRGAP and GYPGKF failed to activate cultured microglia. In addition, hirudin, a specific inhibitor of thrombin could not inhibit the thrombin-stimulated microglial activation suggesting the non-proteolytic activity of thrombin in microglial activation mechanisms. In contrast, thrombin-induced direct neurotoxicity was almost completely blocked by hirudin in neuron-enriched mesencephalic and cortical cultures. PAR-4 agonist peptide (GYPGKF) could mimic the effect of thrombin in neuron-enriched mesencephalic culture. However, the agonist peptides of thrombin receptors such as SFLLRN, TFRGAP and GYPGKF failed to exert a direct neurotoxicity in cortical cultures suggesting cell type specific action of thrombin receptors. Collectively, these results indicate that thrombin can act as a neurotoxin directly and indirectly to cause cell death of cortical and mesencephalic neurons and to induce microglial activation. The present study further suggest that contrary to the thrombin-induced direct toxicity, non-proteolytic activity may mediate thrombin-induced microglial activation.
more초록/요약
Thrombin은 지혈과 상처 치료에 중요한 역할을 하는 복합적인 기능을 가진 단백질이며 생체내에 존재하는 전구체인 prothrombin으로부터 형성된다. Thrombin은 뇌혈관장벽의 파손에 의해서 혈장으로부터 뇌 조직으로 유입될 수 있다. 지금까지 신경세포와 신경교세포의 여러 생리활성에 대한 thrombin의 역할에 관한 많은 연구가 보고되어져 있다. 최근 연구 결과에 의하면 척수의 운동 신경세포와 해마 신경세포를 포함한 많은 종류의 신경세포에서 thrombin이 신경독성물질로 작용한다고 알려져 있다. 또한 thrombin은 마이크로글리아의 활성화를 유도하여 염증반응에 관여하는 여러 싸이토카인의 발현을 촉진 시키기도 한다. 이러한 thrombin의 생체내 다양한 생리 활성은 G-protein-coupled receptor 의 일종인 protease activated receptor (PAR) 를 경유하는 것으로 보고되어져 있다. 본 연구의 목적은 thrombin에 의한 마이크로글리아의 활성화와 신경세포 사멸과정의 기전을 밝히고자 함이다. 면역화학염색법과 생화학적인 분석의 결과 마이크로글리아가 존재할 경우 thrombin에 의해 중뇌 신경세포와 피질 신경세포의 심각한 사멸이 유도되었으며 또한 OX-6 항체를 이용한 면역화학염색결과 피질의 마이크로글리아의 활성화도 매우 증가시키는 것을 관찰할 수 있었다. 그리고 이에 따른 싸이토카인 (TNF-α, IL-1β and IL-6) 들과 iNOS, COX-2 의 발현 또한 매우 증가함을 관찰할 수 있었다. 중뇌 세포배양에서 thrombin에 의해 활성화된 마이크로글리아로부터 얻어진 conditioned media 가 도파민성 신경세포의 사멸을 매우 촉진시키는 것을 관찰 하였다. 그러나, thrombin 수용체인 PAR-1-4의 agonist 펩타이드의 경우 thrombin과는 달리 마이크로글리아의 활성화를 유도하지 못하는 것을 관찰하였다. 또한, thrombin의 억제제인 히루딘 또한 쓰롬빈에 의한 마이크로글리아의 활성화를 억제시키지 못하였다. 이러한 결과들로 미루어 thrombin의 비효소활성이 마이크로글리아의 활성화 과정에서 중요한 역할을 한다는 사실을 알 수 있다. 그와 반대로 마이크로글리아와 독립적으로 일어나는 thrombin에 의한 직접적인 신경세포 사멸의 경우, 히루딘에 의해 세포 사멸이 억제되는 것을 중뇌 신경세포와 피질 신경세포 배양에서 관찰하였다. 중뇌 신경세포의 경우 PAR-4 의 agonist 에 의해 thrombin의 효과가 재현됨을 볼 수 있었다. 그러나, 피질 신경세포의 경우는 어떤 PAR antagoist 에 의해서도 신경세포의 사멸이 일어나지 않는 것을 관찰 하였다. 이러한 결과들을 종합할 때, thrombin은 중뇌와 피질의 신경세포에 직접, 또는 간접적으로 신경독성물질로 작용하며 마이크로글리아의 활성인자로도 작용한다. 본 연구는 또한 thrombin에 의한 마이크로글리아의 활성화는 비효소활성에 의하여 이루어지며 thrombin에 의한 직접적인 신경세포사멸은 thrombin의 효소활성에 의해 이루어진다는 사실을 시사한다.
more목차
TABLE OF CONTENTS
ACKNOWLEGEMENTS = ⅰ
ABSTRACT = ⅱ
TABLE OF CONTENTS = ⅳ
LIST of FIGURES = ⅷ
LIST of TABLE = ⅹ
LIST of ABBREVIATION= xi
Ⅰ. INTRODUCTION = 1
1. Thrombin in the CNS = 1
1.1. Protective role of thrombin in the brain = 1
1.2. Role of thrombin in pathological conditions = 2
1.3. Thrombin inhibitors = 3
2. The role of thrombin receptors = 5
2.1. Protease activated receptors (PARs) = 5
2.2. Thrombin receptor signaling = 6
2.2.1. PAR-1 = 6
2.2.2. PAR-3 and -4 = 7
2.3. Role of PARs in the brain = 7
2.4. Non-proteolytic activity of thrombin = 8
3. Microglia and their role in the CNS = 9
3.1. Microglia = 9
3.2. Characteristics of microglial activation = 10
3.2.1. Morphological change = 10
3.2.2. Change of surface antigen = 11
3.2.3. Expression of cytokines and growth factors = 11
3.3. Protective roles of microglia = 12
3.4. Deliterous roles of microglia in pathogenic conditions = 13
3.4.1. Reactive oxygen species = 13
3.4.2. Pro-inflammatory cytokines = 15
3.4.3. Chemokines = 16
3.5. Inflammation in PD brain = 16
3.6. Inflammation in AD brain = 17
4. Aims of study = 18
Ⅱ. MATERIALS AND METHODS = 19
1. Chemicals = 19
2. Neuron-enriched mesencephalic cultures = 19
3. Cortical microglia cultures = 20
4. Neuron-enriched cortical cultures = 21
5. Co-cultures of cortical microglia and neurons = 21
6. Co-cultures of cortical microglia and mesencephalic neurons = 22
7. Injection of thrombin into the cerebral cortex = 22
8. Pretreatment with L-NAME and NS-398 = 23
9. Immunocytochemistry and Immunohistochemistry = 23
10. Immunofluorescence double-labeling = 24
11. Terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick-end labeling (TUNEL) assay = 25
12. Measurement of dopamine uptake = 26
13. Determination of NO = 27
14. Quantification of TNF-α release = 27
15. Western Blot analysis = 27
16. Thrombin enzyme assay (Chromogenic assay) = 28
17. Reverse transcription polymerase chain reaction (RT-PCR) = 28
18. Measurement of cell death (Live and dead cell assay) = 30
19. Statistical analysis = 30
Ⅲ. RESULTS = 32
Part A. Thrombin-induced microglia activation increases dopaminergic neuronal cell death = 32
1. Thrombin induces cell death of dopaminergic neurons in microglia-neuron co-cultures = 32
2. Conditioned media (CM) derived from thrombin-stimulated cortical microglia induces degeneration of dopaminergic neurons in mesencephalic cultures = 35
3. Thrombin induces expression of the proinflammatory cytokines, iNOS and COX-2 in cultured cortical microglia = 37
4. Inhibition of microglial activation by MAPK pathway inhibitors rescues dopaminergic neurons = 39
5. p38-MAPK and JNK activation contributes to cell death of dopaminergic neurons in mesencephalic cultures treated with CM of thrombin-activated microglia = 41
6. Non-proteolytic actions of thrombin contribute to activation of microglia = 43
Part B. Thrombin induces microglia activation and cortical neuronal cell death in vivo and in vitro via protease activated receptor-independent pathway = 49
1. Thrombin induces neuronal cell death in rat brain cortex in vivo = 49
2. Thrombin induces microglia activation and produces proinflammatory cytokines, iNOS and COX-2 in the cortex in vivo = 52
3. Neurotoxic actions of iNOS and COX-2 on degeneration of cortical neurons in vivo = 54
4. Thrombin induces cortical neuronal cell death in co-culture of cortical microglia and neurons = 55
5. Thrombin activates microglia through non-proteolytic activity and PAR-independent pathway = 59
Part C. Role of proteolytic activity of thrombin and thrombin receptors in thrombin-induced direct neurotoxicity = 65
1. Thrombin exerts direct toxicity against dopaminergic neurons in rat mesencephalic culture = 65
2. Thrombin-induced neurotoxic actions on dopaminergic neurons in mesencephalic cultures are not mediated by PAR-1 = 72
3. Direct toxicity of thrombin can be blocked by hirudin in neuron-enriched cortical cultures 72
Ⅳ. DISCUSSIONS = 74
Part A. Thrombin induces dopaminergic neuronal cell death by activation of microglia = 74
Part B. Thrombin induces microglia activation and cortical neuronal cell death in vivo and in vitro via protease activated receptor-independent pathway = 80
Part C. Mechanisms of thrombin-induced direct neurotoxicity: Role of proteolytic activity of thrombin and thrombin receptors = 85
Ⅴ. SUMMARY AND CONCLUSION = 87
Ⅵ. BIBLIOGRAPHY = 88
국문초록 = 105
