Title Page

Contents

ABSTRACT 10

Ⅰ. INTRODUCTION 12

Ⅱ. MATERIALS AND METHODS 16

1. Ethics approval 16

2. Study participants 16

3. Extracting RNA from peripheral blood mononuclear cells 17

4. Quantitative reverse transcription polymerase chain reaction 18

5. Mouse α-synuclein pre-formed fibril production and stereotaxic surgery 20

6. Immunoblotting 21

7. Immunohistochemistry 23

8. Sectioning of mouse brain and immunofluorescence analysis 24

9. Behaviour tests 25

10. Flow cytometry analysis 26

11. Statistical analysis 29

Ⅲ. RESULTS 31

1. Demographic overview of participants 31

2. Elevation of PSMB8 mRNA and protein levels in the peripheral blood and central nervous system of Parkinson's disease patients 33

2.1. Selective upregulation of PSMB8 at both mRNA and protein levels in PBMC of patients with PD 33

2.2. Evidence related to expression of PSMB8 protein in central nervous system 38

3. Immunoproteasome correlates with Parkinson's disease severity and its roles as a potential biomarker 38

4. Establishing Parkinson's disease mouse model through unilateral striatal injection of α-Synuclein pre-formed fibril 40

5. Distinct temporal upregulation of PSMB8 in PFF mouse model of PD 42

6. Independent expression of three catalytic subunits in dopaminergic neurons and microglia within the substantia nigra 45

7. Distinct elevation of immunoproteasome in the dopaminergic neurons and microglia confirmed via flow cytometry. 48

Ⅳ. DISCUSSION 54

Ⅴ. REFERENCES 60

KOREAN ABSTRACT 64

SUPPLEMENTARY 66

Table 1. The sequence of primers used for Real-time PCR 19

Table 2. Clinical features of PD patients and healthy controls included in this study 31

Table 3. Results of ANCOVA for each immunoproteasome related target between healthy control and each patient group. 35

Table S4. List of chemicals used in this study 66

Table S5. List of antibodies used in this study 71

Table S6. List of instruments and devices used in this study 74

Figure 1. Structures and function of ubiquitin-proteasome system 14

Figure 2. Research Scheme 20

Figure 3. Immunoproteasome mRNA in the of the Parkinson's disease (PD) group or divided subgroups compared with healthy controls (HC). 34

Figure 4. The expressed protein levels of immunoproteasome subunits in peripheral blood mononuclear cells 36

Figure 5. The expression of PSMB8 protein in the brains of patients and control 37

Figure 6. Correlation assessments between PSMB10/8 and disease severity and receiver operating characteristic analysis. 39

Figure 7. Evolution of α-Synuclein pathology and behavioral impairment in the PD mouse model induced by unilateral striatal injection 42

Figure 8. Temporal alteration of immunoproteasome catalytic subunits in Parkinson's disease mouse model 44

Figure 9. Distinct expression pattern of Immunoproteasome catalytic subunits in the substantia nigra at early timepoint 45

Figure 10. Distinct expression pattern of Immunoproteasome catalytic subunits in the substantia nigra at late timepoint 46

Figure 11. Expression of immunoproteasome in microglia at late time point of 90 days post-injection 47

Figure 12. Representative gating strategies in flow cytometry 49

Figure 13. Differential expression of immunoproteasomes in dopaminergic neurons and microglia via flow cytometry 53

Figure 14. Landscape of immunoproteasome contribution on PD pathogenesis 59