Roles of FcγRIIb signaling pathways in the pathogenesis of Parkinson’s disease
- 주제(키워드) α-Synuclein fibrils , Prion-like propagation , FcγRIIB , SHP-1 , SHP-2 , c-Src , Microglia phagocytosis , Neuron , Parkinson’s disease.
- 발행기관 아주대학교
- 지도교수 박상면
- 발행년도 2019
- 학위수여년월 2019. 2
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000028551
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
α-Synuclein (α-Syn) has been considered to be a key player of the pathogenesis of PD, and recent reports that prion-like propagation of misfolded α-syn released from neurons may play an important role in the progression of PD have led to increased attention to the studies elucidating the roles of extracellular α-syn in the CNS. Extracellular α-syn has also been reported to regulate microglial inflammatory response including phagocytosis. Here, I demonstrate that how α-syn fibrils inhibits microglial phagocytosis to take advantage of immune complexes-induced phagocytosis model. α-syn fibrils bound to FcγRIIB on microglia, inducing SHP-1 activation, further inhibiting microglial phagocytosis. Further, I show that FcγRIIB expressed in neurons functions as a receptor for α-syn fibrils and mediates cell-to-cell transmission of α-syn. SHP-1 /-2 and c-Src are activated downstream by α-syn fibrils through FcγRIIB and play an important role in cell-to-cell transmission of α-syn. Also, taking advantage of a newly developed co-culture system, I show that cell-to-cell transmission of α-syn induces intracellular Lewy body-like inclusion body formation and that the FcγRIIB/SHP-1/-2/c-src signaling pathway is involved in it. Therefore, the FcγRIIB-SHP-1/-2/c-src signaling pathway may be a novel therapeutic target for the progression of PD and an in vitro model system provides an efficient tool for further high throughput screening that can be used for developing a therapeutic intervention in PD.
more목차
I. ITRODUCTION 1
THEME I. FCΓRIIB MEDIATES THE INHIBITORY EFFECT OF Α-SYN FIBRILS ON MICROGLIA PHAGOCYTOSIS 1
THEME II. PRION-LIKE PROPAGATION OF Α-SYN IS REGULATED BY THE FCΓRIIB/SHP-1/-2/C-SRC SIGNALING PATHWAY IN NEURON 3
II. METERIALS AND METHODS 6
1. Reagent and antibody 6
2. Constructs 6
3. Purification of recombinant α-syn and preparation of α-syn fibrils 7
4. Thioflavin T Assay and Tranmission Electoron Microscopy (TEM) 8
5. Generation of stable cell lines 8
6. Cell culture 9
7. In vitro phagocytosis assay 10
8. Preparation of ICs (immune complex) 10
9. Preparation of Brain lysate 11
10. Western blot 11
11. Coimmunoprecipitation 12
12. Quantitative Real-time RT-PCR 12
13. In vitro cell to cell transmission assay using dual chamber 13
14. Coculture assay 13
15. Confocal microscopy 13
16. Statistical analysis 14
III. RESULTS 15
THEME I. FCΓRIIB MEDIATES THE INHIBITORY EFFECT OF Α-SYN FIBRILS ON MICROGLIA PHAGOCYTOSIS 15
1. α-Syn fibrils inhibits microglial phagocytosis in a dose-dependent manner. 15
2. Immune Complexes (ICs)-induced phagocytosis is also inhibited by α-syn fibrils. 19
3. α-Syn fibrils inhibits ICs-induced signaling pathway by SHP-1 activation. 22
4. SHP-1 is essential for the inhibition of microglial phagocytosis by α-syn fibrils. 25
5. α-Syn fibrils activates SHP-1 by interacting with FcγRIIB. 28
THEME II. PRION-LIKE PROPAGATION OF Α-SYN IS REGULATED BY THE FCΓRIIB/SHP-1/-2/C-SRC SIGNALING PATHWAY IN NEURON 34
6. α-Syn fibrils bind to human FcγRIIB 34
7. FcγRIIB downstream signaling is involved in cell-to-cell transmission of α-syn 37
8. α-Syn fibrils induce the phosphorylation of SHP-1 and SHP-2 by interacting with FcγRIIB 42
9. SHP-1/-2 expressed in neurons mediate cell-to-cell transmission of α-syn 48
10. FcγRIIB-SHP-1/2 Downstream Signaling Is Involved in Lipid Raft-Dependent Endocytosis 53
11. Development of an in vitro model system to quantify the formation of Lewy body-like inclusions by cell-to-cell transmission of α-syn 56
12. FcγRIIB-SHP-1/-2 signaling pathway mediates the formation of Lewy body-like inclusion induced by cell-to-cell transmission of α-syn fibrils 61
13. α-Syn fibrils induced c-src activity through FcγRIIB-SHP-1/2 pathway. 65
14. c-Src mediate cell-to-cell transmission of α-Syn. 68
15. c-Src mediates the formation of Lewy body-like inclusion induced by cell-to-cell transmission of α-syn fibrils 70
16. c-Src is also involved in the release of α-syn and degradation of α-syn through the autophagy. 73
IV. DISCUSSION 76
THEME I. FCΓRIIB MEDIATES THE INHIBITORY EFFECT OF Α-SYN FIBRILS ON MICROGLIA PHAGOCYTOSIS 76
THEME II. PRION-LIKE PROPAGATION OF Α-SYN IS REGULATED BY THE FCΓRIIB/SHP-1/-2/C-SRC SIGNALING PATHWAY IN NEURON 77
V. CONCLUSION 83
REFERENCE 84
국문 요약 95
