Title Page

Contents

Abbreviations 7

Abstract 10

Ⅰ. Introduction 11

Ⅱ. Methodology 14

1. Chemicals and instruments 14

2. Methods 14

2.1. Expression of recombinant human α-synuclein in E. coli BL21 (DE3) 14

2.2. Human α-synuclein recombinant protein purification 14

2.3. SDS-PAGE analyses 17

2.4. Human sample preparations and ethical statement 17

2.5. Neuronal exosome isolation from serum 18

2.6. Immunoblotting 19

2.7. α-synuclein RT-QuIC assay 20

2.8. Statistical analysis 20

Ⅲ. Results 22

1. Comparison between manual and AKTA human α-synuclein purification 22

2. Confirmation of the recombinant α-synuclein seeding ability by CSF RT-QuIC 23

3. Relationship between OM RT-QuIC results and olfactory function in PD patients 26

4. Confirmation of neuronal exosome isolation by immunoblotting 30

Ⅳ. Discussion 32

Ⅴ. Conclusion 34

Ⅵ. Supplementary 35

Ⅶ. References 39

Abstract 43

Table 1. Demographics, clinical characteristics, and OM RT-QuIC results 27

Table 2. Chemicals used for this study 35

Table 3. Antibody used in this study 37

Table 4. Instruments used for this study 37

Figure 1. The principle of RT-QuIC assay 12

Figure 2. Culture, expression, and purification of recombinant human α-synuclein scheme 15

Figure 3. Neuronal exosome isolation scheme 18

Figure 4. Protein purification results 23

Figure 5. Optimizing the condition of CSF RT-QuIC assay 24

Figure 6. CSF RT-QuIC assay 25

Figure 7. Relationship between OM RT-QuIC results and olfactory function 28

Figure 8. Immunoblotting confirmation of neuronal exosome isolation from serum 30

Figure 9. pSer129 α -synuclein levels in neuronal exosome 31