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DJ-1, a Parkinson’s Disease-associated Gene, Prevent Excessive Glial Inflammatory Responses through Interaction with SHP-1

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dc.contributor.author김, 종현-
dc.date.accessioned2014-11-10T04:04:52Z-
dc.date.available2014-11-10T04:04:52Z-
dc.date.issued2014-
dc.identifier.urihttp://repository.ajou.ac.kr/handle/201003/10858-
dc.description.abstractParkinson’s disease (PD) is a progressive neurodegenerative movement disorder caused by the death of dopaminergic neurons in the substantia nigra. Importantly, altered astrocyte and microglial functions could contribute to neuronal death in PD. In this study, I demonstrate a novel mechanism by which DJ-1 (PARK7), an early onset autosomal recessive PD gene, negatively regulates inflammatory responses of astrocytes and microglia by facilitating the interaction between STAT1 and its phosphatase, SHP-1 (Src-homology 2-domain containing protein tyrosine phosphatase-1). Astrocytes and microglia cultured from DJ-1-knockout (KO) mice exhibited increased expression of inflammatory mediators and phosphorylation levels of STAT1 (p-STAT1) in response to interferon-gamma (IFN-g) compared to cells from wild-type (WT) mice. DJ-1 deficiency also attenuated IFN-g-induced interactions of SHP-1 with p-STAT1 and STAT1, measured 1 and 12 h after IFN-g treatment, respectively. Notably, DJ-1 bound to SHP-1 independently of IFN-g, whereas the interactions of DJ-1 with p-STAT1 and STAT1 were dependent on IFN-g. Similar results were obtained in brain slice cultures, where IFN-g induced much stronger STAT1 phosphorylation and inflammatory responses in KO slices than in WT slices. Moreover, IFN-g treatment induced neuronal damage in KO slices. Collectively, these findings suggest that DJ-1 may function as a scaffold protein that facilitates SHP-1 interactions with p-STAT1 and STAT1, thereby preventing extensive and prolonged STAT1 activation. Thus, the loss of DJ-1 function may increase the risk of PD by enhancing brain inflammation.-
dc.description.abstract파킨슨병 (Parkinson's disease) 은 중뇌 흑질 부위의 도파민 생성 신경세포가 사멸하여 발생하는 운동성 퇴행성뇌질환이다. 본 연구에서는 조기에 발병하는 파킨슨병 유전자인 DJ-1 (PARK7) 이 파킨슨병의 위험인자로 알려진 뇌염증 반응을 조절할 가능성을 확인하였다. DJ-1 을 제거한 (DJ-1 KO) 생쥐로부터 배양한 성상세포 (Astrocytes) 와 소교세포 (Microglia) 에서 DJ-1이 온전한 (WT) 생쥐로부터 배양한 세포들보다 interferon-gamma (IFN-g) 에 의한 염증매개물질들의 발현과 STAT1 의 인산화가 증가 되었다. IFN-g 처리 후, p-STAT1 과 STAT1 은 탈인산화효소인 SHP-1 과 1시간 또는 12 시간에 결합력이 증가하는데, DJ-1 이 없는 경우 이러한 결합력이 줄어드는 것을 확인하였다. 특히, DJ-1 과 SHP-1 은 IFN-g 처리와 관계없이 결합하며, p-STAT1 이나 STAT1 과는 IFN-g 의존적으로 결합하는 것을 확인하였다. 또한, 추가 실험을 통해 DJ-1 과 p-STAT1/STAT1 과의 결합을 SHP-1 에 의존적으로 일어남을 확인하였다. 이러한 결과는 대뇌 피질 절편 배양 (Cortical slice culture) 실험에서도 얻을 수 있었다. DJ-1 KO 절편에 IFN-g 를 처리하면 WT 절편에 비해 STAT1 의 인산화와 염증반응이 더 강하게 일어나며, WT 절편에 비해 신경세포의 사멸이 유도됨을 확인했다. 종합적으로, 이러한 결과들은 DJ-1 이 SHP-1 과 p-STAT1/STAT1 간의 결합을 촉진하여 STAT1 이 오랫동안 과도하게 활성화되는 것을 막는 역할을 하는 scaffold 단백질로 기능을 할 가능성을 의미한다. 그러므로, DJ-1 기능 소실은 뇌 염증반응의 과도한 활성화를 유발하고, 이로 인해 파킨슨병의 위험요소가 증가될 수 있을 것으로 생각된다.-
dc.description.tableofcontentsABSTRACT i

TABLE OF CONTENTS iii

LIST OF FIGURES vi

LIST OF TABLE viii

ABBREVIATION ix

I. INTRODUCTION 1

A. Parkinson's disease (PD) 1

B. Genes associated with autosomal recessive PD 2

1. DJ-1 (PARK7) 2

2. Parkin (PARK2) 4

3. PINK1 (PAKK6) 5

C. Glial cells 6

1. Microglia 6

2. Astrocytes 7

D. Brain inflammation 8

1. Brain inflammation 8

2. Interferon-gamma (IFN-g) 9

3. JAK/STAT pathway 10

E. Negative regulators of JAK/STAT pathway 11

1. Protein tyrosine phosphatases (PTPs): SHP-1 and SHP-2 11

2. SOCS 13

3. PIAS 14

F. Aims of this study 15

II. Materials and methods 16

1. DJ-1-deficient mice 16

2. Cell culture 16

3. Organotypic cortical slice cultures 17

4. Preparation of SHP-1-knockdown BV2 cells 17

5. Quantitative real-time polymerase chain reaction 17

6. Western blot analysis 18

7. Cytosolic and nuclear fractionation 19

8. Immunoprecipitation 20

9. Immunocytochemistry 20

10. Proximity ligation assay (PLA) 21

11. Transfection 21

12. Plasmids 21

13. Live/Dead assay 22

14. Statistical analysis 22

III. RESULTS 23

1. Anti-inflammatory functions of DJ-1 in IFN-g-treated astrocytes and microglia 23

2. DJ-1 deficiency decreases the interactions of SHP-1 with p-STAT1 and STAT1 32

3. DJ-1 interacts with SHP-1, STAT1, and p-STAT1 38

4. The interactions of DJ-1 with SHP-1, p-STAT1, and STAT occur in both nucleus and cytosol 43

5. SHP-1 deficiency attenuates the interaction of DJ-1 with STAT1 and p-STAT 45

6. DJ-1 KO increases IFN-g-induced inflammatory responses and neuronal death in IFN-gtreated brain slices 48

IV. DISCUSSION 52

V. SUMMARY AND CONCLUSION 59

REFERENCES 60

국문요약 84
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dc.language.isoen-
dc.titleDJ-1, a Parkinson’s Disease-associated Gene, Prevent Excessive Glial Inflammatory Responses through Interaction with SHP-1-
dc.title.alternativeDJ-1과 SHP-1의 상호작용을 통한 신경교세포의 과도한 면역반응 억제-
dc.typeThesis-
dc.identifier.urlhttp://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000015977-
dc.subject.keywordParkinson's disease-
dc.subject.keywordDJ-1-
dc.subject.keywordSHP-1-
dc.subject.keywordSTAT1-
dc.subject.keywordBrain inflammation-
dc.subject.keyword파킨슨병-
dc.subject.keyword뇌 염증반응-
dc.description.degreeDoctor-
dc.contributor.department대학원 의생명과학과-
dc.contributor.affiliatedAuthor김, 종현-
dc.date.awarded2014-
dc.type.localTheses-
dc.citation.date2014-
dc.embargo.liftdate9999-12-31-
dc.embargo.terms9999-12-31-
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