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파킨슨병 유전자 DJ-1에 의한 성상세포 기능 조절 연구

Regulation of Astrocyte Functions by a Parkinson's Disease Gene, DJ-1

초록/요약

Parkinson’s disease (PD) is a neurodegenerative motor disease, accompanied by loss of dopaminergic neurons in the substantia nigra. PD genes have been studied to reveal molecular mechanisms of onset and progression of PD. In this study, I investigated the effect of a PD gene, DJ-1, on astrocyte function since astrocytes as the most abundant cells in the brain play diverse roles for well-being and function of the brain. In the first part, I investigated behavior of astrocytes in ATP-injured striatum. In response to injury, DJ-1 KO astrocytes did not undergo proper gliosis: less increase in expression of GFAP, nestin, and GDNF, and less hypertrophic. In addition, damage areas in DJ-1 KO brain were slowly recovered with astrocytes and TH-positive neurites compared with WT brain. As an underlying mechanism, I found that DJ-1 deficiency attenuateded activation of STAT3, a transcription factor that regulates expression of GFAP and nestin. In the second part of the study, I investigated anti-inflammatory roles of astrocytes regulated by DJ-1. DJ-1 deficient astrocytes less efficiently reduced microglial expression of proinflammatory mediators such as iNOS and TNF-. DJ-1 KO astrocytes compared with WT astrocytes less produced anti-inflammatory prostaglandin, prostaglandin D2 (PGD2), through attenuated expression of lipocalin-type PGD2 synthase (L-PGDS). DJ-1 regulates protein stability of Sox9, a transcription factor that regulates L-PGDS expression. Thus, in the absence of DJ-1, Sox9 ubiquitination was increased and the protein was degraded. Taken together, these results indicate that DJ-1 regulated numerous functions of astrocytes. Therefore, DJ-1 deficiency may affect onset and/or progression of PD due to defects in astrocyte function in intact and injured brain.

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목차

INTRODUCTION . 1
A. Parkinson's disease (PD) 1
B. Genes associated with PD . 2
1. DJ-1 (PARK7) 2
2. Other genes . 3
C. Function of astrocytes in the normal brain . 6
D. Function of astrocytes in the injured brain . 7
1. Reactive astrocytes : intermediate filaments (Ifs) and morphological features . 7
2. Reactive astrocytes : function of astrogliosis in neural protection . 8
3. Reactive astrocytes : function of astrogliosis for regeneration and repair . 9
4. Reactive astorycytes : functin of astrogliosis for regulation of inflammation . 11
E. Astrocyte dysfunction in neurodegenerative disease 12
F. Aim of this study 13
II. Materials and methods 15
1. Ethics statement . 15
2. DJ-1 deficient mice . 15
3. Animal MRI 15
4. Stereotaxic surgery and drug injection . 16
5. Tissue preparation . 16
6. Immunohistochemistry 17
7. Histological quantification 18
8. Organotypic cortical slice cultures 18
9. Cell culture 19
10. Western blot analysis . 19
11. Immunoprecipitation 21
12. Quantitative real-time PCR. 21
13. Reverse transcriptase PCR 22
14. DNA constructs . 23
15. Transfection 23
16. Proximity ligation assay (PLA) 23
17. Small interfering RNAs (siRNA) 24
18. Prostaglandin D2 (PGD2) ELISA . 25
19. Inhibition of protein synthesis by cycloheximide 25
20. Inhibition of Ubiquitination by MG132 25
21. Measurement of nitric oxide 25
22. Statistical analysis . 26
III. RESULTS 27
Part A. DJ-1, (PARK7), plays critical roles in astrogliosis by regulating of the pSTAT3 signaling pathway for tissue repair after brain injury 27
1. Repair of brain injury was attenuated in DJ-1 KO mice 27
2. DJ-1 deficiency causes a defect of astrogliosis . 30
3. Reduction of astrocyte derived GDNF expression and attenuated restoration of dopaminergic processes in the DJ-1 KO brain 41
4. DJ-1 rescued insufficient astrogliosis in DJ-1 KO brain slice cultures. 47
5. STAT3 activation (tyrosine phosphorylation of STAT3, pSTAT3) was attenuated in DJ- 1 KO slices 50
Part B. DJ-1 exerts the anti-inflammatory effects of astrocytes through stabilization of Sox9, a transcription factor that regulates lipocalin-type prostaglandin D2 synthase (L-PGDS) expression. 52
1. DJ-1 deficiency caused a defect of anti-inflammatory function of astrocytes. 52
2. DJ-1 deficiency caused a defect of PGD2 expression by attenuation of L-PGDS in astrocytes. 57
3. DJ-1 deficiency caused a defect of Sox9 protein expression by regulation of ubiquitination in astrocytes. 65
IV. DISCUSSION 73
V. SUMMARY AND CONCLUSION. 85
REFERENCES. 86
국문요약 . 109

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