MPTP 를 이용한 파킨슨병 동물 모델에서의 케나비노이드 수용체와 바닐로이드 수용체 기능 규명
Studies on the role of CB and TRPV1 receptor in MPTP model of Parkinson's Disease
- 주제(키워드) Parkinson’s disease , Dopaminergic neuron , Cannabinoid receptor , Transient receptor potential vanilloid subtype 1 , neuroinflammation
- 발행기관 아주대학교
- 지도교수 백은주/ 진병관
- 발행년도 2010
- 학위수여년월 2010. 8
- 학위명 박사
- 학과 및 전공 일반대학원 신경과학기술과정
- 실제URI http://www.dcollection.net/handler/ajou/000000010866
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Parkinson’s disease (PD) is characterized by the degeneration of nigrostriatal dopaminergic (DA) neurons. Mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) exhibit glial activation-derived oxidative stress and inflammation, damaged blood-brain barrier (BBB), infiltrated peripheral immune cells and nigrostriatal DA neuronal damage, and thus serve as an experimental model of PD. Increasing evidence indicates that agonists of cannabinoid (CB) receptor and transient receptor potential vanilloid subtype 1 (TRPV1) have been shown to reduce the production of inflammatory mediators. In addition, recent studies have suggested that these receptors are implicated with the pathogenesis of neurodegenerative disease, such as Alzheimer’s disease, Parkinson’s disease and Huntington’s disease. The purpose of this study was to investigate the role of CB receptor and TRPV1 on nigrostriatal pathway in MPTP mouse model of PD. Immunohistochemical and biochemical evidence demonstrated that MPTP injection resulted in a significant loss of DA neurons and microglial activation in the nigrostriatal pathway. Western blot analysis and double-label immunohistochemistry show that the translocation of cytosolic proteins (p47phox and Rac1) to the membrane, and p47phox expression of NADPH oxidase in microglia in the SN in vivo, indicating the activation of NADPH oxidase. Reactive oxygen species (ROS) production and oxidative damage, assessed by hydroethidine histochemistry and western blotting, were observed in the SN area in which degeneration of DA neurons occurred. In parallel, RT-PCR analysis and immunohistochemical results showed that glial activation-derived proinflammatory mediators were increased in the MPTP-treated SN. The passage of FITC-labeled albumin (FITC-LA) from blood into SN also showed the MPTP-induced damage of the brain blood barrier (BBB). Additional immunohistotochemical staining revealed peripheral immune cell (T cell, B cell, macrophage and neutorphil) infiltration in the SN and STR. However, treatment with cannabinoids (HU210 and WIN55,212-2) prevents MPTP-induced degeneration of nigrostriatal DA neurons by inhibiting transient expression of proinflammatory cytokines and inducible nitric oxide synthase (iNOS); and attenuating microglial NADPH oxidase activation, reactive oxygen species/reactive nitrogen species production, and consequent oxidative damage. All of these neuroprotective effects were reversed by treatment with CB1 antagonist, AM251 and SR14716a, indicative of CB1 receptor mediation. Moreover, treatment with WIN55,212-2 (agonist of CB1/2) and JWH-133 (selective CB2 agonist) increased survival of DA neurons in the SN, and their fibers in the STR. This neuroprotection is accompanied by preventing of glial activation-derived proinflammatory mediators, BBB disruption and peripheral immune cell infiltration. These in vivo effects were ameliorated by treatment with CB2 antagonists AM630, suggesting direct involvement of CB2 in neuroprotection. Additional study demonstrated that treatment with capsaicin (CAP), TRPV1 agonist, also prevented degeneration of nigrostriatal DA neurons, increased striatal dopamine levels and improved motor function. This neuroprotection afforded by CAP was associated with NADPH oxidase-derived ROS production, reduced expression of pro-inflammatory cytokines by activated microglia. This neuroprotective effects were reversed by treatment with TRPV1 antagonist capsazepine (CZP) and iodo-resinoferatoxin (I-RTX), indicative of TRPV1 receptor mediation. Collectively, these results suggest that cannabinoid and vanilloid system may be beneficial for the treatment of neurodegenerative diseases, such as PD, that are associated with glial activation-derived oxidative stress and proinflammatory mediators, BBB leakage and peripheral immune cell infiltration.
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ABSTRACT ••••••••••••••••••••••••••••i
TABLE OF CONTENTS •••••••••••••iv
LIST OF FIGURES ••••••••••••••x
LIST OF TABLES •••••••xii
ABBREVIATION •••••••xiii
1. Parkinson’s disease (PD)•••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••1
2. Pathogeneis of Parkinson’s disease ••••••1
3. Experimental models of Parkinson’s disease ••2
3.1. MPTP/MPP+ model of PD••••••••••••3
3.2. 6-OHDA model of PD••••••••••••••3
3.3. Rotenone and Paraquat model of PD••••••4
3.4. Genetic model of PD•••••••••••••••••••••••5
4. Involvement of neuroinflammation in Parkinson’s disease ••••••••••••••••••••••••••••••••••••••••5
4.1. Microglial activation in Parkinson’s disease •••6
4.2. Oxidative stress and proinflammatory cytokines in Parkinson’s disease •••••••••••••••••••••7
4.3. Astrocytes in the brain ••••••8
4.4. Dysfunction of blood brain barrier in PD •••9
4.5. Infiltration of peripheral immune cells in PD •••10
5. Characteristics of cannabinoid receptor •••11
5.1. Cannabinoid receptor type 1 (CB1) •••12
5.2. Cannabinoid receptor type 2 (CB2) ••13
6. Characteristics of Transient Receptor Potential Vanilloid Subtype 1 (TRPV1) •••••••••••••••14
7. Roles of CB and TRPV1 in the CNS ••••17
7.1. Neuroprotective effect of CB1 in CNS••••••18
7.2. Neuroprotective effects of CB2 in CNS••••19
7.3. Neuroprotective effect of TRPV1••••20
8. Endocannabinoid/endovanilloid system implicated with PD•••••••••••••••••••••••••••••••••••••21
9. Aims of this study •••••22
Ⅱ. MATERIALS AND METHODS••••23
A. MATERIALS••••••23
1. Chemicals••••23
2. Aniamals and drugs treatment•••••••••••••23
3. Neuron-enriched mesencephalic cultures and drugs treatment••••••••••••••••••••••••••••••••••••24
4. Mesencephalic microglia cultures••••••25
B. METHODS•••••25
1. Measurement of MPTP and MPP+ levels in the striatum•••••••25
2. Tissue preparation and immunostaining•••••26
3. Stereological cell counts••••••27
4. Densitometric analysis•••28
5. Rotarod test••••••29
6. Measurement of dopamine levels in striatum•••••29
7. Immunofluoxrescence double labelling••••30
8. In situ detection of O2- and O2--derived oxidants•••31
9. Western bolt assay••••31
10. Detection for protein carbonylation •••••32
11. RT (Reverse-transcription) PCR •••••33
12. FITC-linked albumin assay •••••35
13. Statistical analysis ••••35
Ⅲ. RESULTS•••••••••37
Part A. Cannabinoid receptor type 1 (CB1) protects nigrostriatal dopaminergic neuron from MPTP neurotoxicity by inhibiting microglial activation•••••••••••••••37
1. WIN55,212-2 and HU210 protect nigrostriatal DA neurons from the MPTP neurotoxicity via CB1 receptor activation in vivo. •••••••••••••••••••••••••••••••••37
2. The motor deficits and dopamine depletion-induced MPTP attenuated by CB1 receptor activation in vivo. •••••44
3. WIN55,212-2 and HU210 do not alter the metabolism of MPTP to MPP+.••••••••••••••••45
4. The activation of CB1 receptor inhibits the microglial activation and the ROS production in SN. •••••45
5. The neurotoxicity of MPTP was ablated by CB1 receptor activation through inhibition of NADPH oxidase-mediated production of ROS by activated microglia. •••••50
6. Effects of CB1 activation on the MPTP-induced oxidative damages••••••••••••••••••••••••53
7. Blockade of the expression of proinflammatory cytokine and iNOS by CB1 receptor activation. ••••••54
8. Effectiveness of fluoxetine against microglia-derived neurotoxicity•••••••••••••••••••••••••59
Part B. CB2 prevents BBB leakage and peripheral immune cell infiltration and protects nigrostriatal dopaminergic neurons in the MPTP model of Parkinson’s disease. •••••••••••••••63
1. WIN55,212-2 and JWH-133 rescue nigrostriatal DA neurons from the MPTP neurotoxicity via CB2 receptor activation in vivo. •••••63
2. Blockade of microglial activation and production of proinflammatory cytokines by CB2 activation ••••••68
3. Ablation of astroglial activation and MPO expression by CB2 activation. ••••••••••••••••74
4. Effects of CB2 agonists on the MPTP-induced BBB leakage in the SN••••75
5. Effects of WIN55,212-2 and JWH-133 on the MPTP-induced infiltration of peripheral immune cells in the SN••••••77
6. Effects of CB2 receptor activation on the MPTP-induced expression of chemokines•••••80
Part C. TRPV1 protects nigrostriatal dopaminergic neurons in the MPTP model of Parkinson’s disease. ••••••84
1. TRPV1 contributes to survival of nigrostriatal DA neurons in MPTP mousneurotoxicity via TRPV1 receptor activation in vivo. ••••••••••84
2. TRPV1 increases striatal dopamine levels and improves motor behavior in MPTP mice•••90
3. TRPV1 agonist does not alter the metabolism of MPTP to MPP+.•••••••••••••••••••••••••91
4. TRPV1 inhibits microglial activation in the SN in vivo. ••••••••••••••••••••••••••••••••••••93
5. TRPV1 attenuates MPTP-induced oxidant production and oxidative stress via microglial NADPH oxidase••95
6. TRPV1 inhibits MPTP-induced expression of IL-1β, TNF-α and iNOS. •••••••••••••••••98
7. TRPV1 inhibits MPTP-induced astroglial activation and MPO expression in the SN in vivo. ••••••101
8. Effectiveness of capsaicin against MPP+-derived neurotoxicity•••••••••••••••••••••••••••104
Ⅳ. DISCUSSION••••••106
Part A. Neuroprotective role of CB1 via inhibition of oxidative stress and inflammation in MPTP model of PD. ••••106
Part B. Neuroprotective role of CB2 via inhibition of BBB leakage and infiltration of immune cells in MPTP model of PD. •••••112
Part C. Neuroprotective role of TRPV1 via inhibition of oxidative stress and inflammation in MPTP model of PD. ••••••116
Ⅴ. CONCLUSION•••••••••••••••120
REFERENCES•••••••••121
APPENDIX•••••••••166
1. Expression of CB1/2 and TRPV1 in vivo and in vitro•••••••••166
2. MPTP induced in vivo neurotoxicity on DA neurons in the nigrostriatal system. •••••••167
3. MPTP induces microglial activation and expression of proinflammatory cytokines in the SN•••••168
4. MPTP-induced DA neuronal death increased FIT-LA leakage and peripheral immune cells intilfration in the SN. •••••170
5. MPTP and MPP+ levels (μg/mg protein) in the striata of C57 BL/6 mice•••••••••••••••172
6. The Effects of WIN55,212-2 on MPTP-treated nigrostrital DA system in vivo •••••••173
7. The Effects of HU210 on MPTP-treated nigrostrital DA system in vivo. ••••••••••••••174
8. The Effects of JWH-133 on MPTP-treated nigrostrital DA system in vivo. •••••••••••175
9. The Effects of Capsaicin on MPTP-treated nigrostrital DA system in vivo. •••••••••••176
국문요약 •••••••••••••177
