The Pathogenic Roles of S100A8, S100A9 and Their Interaction with SFPQ in Parkinson’s Disease = 파킨슨병에서 S100A8 및 S100A9 의 병원성 역할과 SFPQ 와의 상호 작용

다국어 초록 (Multilingual Abstract)

Parkinson’s disease (PD) refers to a neurodegenerative disorder wherein dopaminergic neurons (DA) in the brain are insolvent or damaged selectively. The pathogenesis of PD is unknown but it is likely to include an intricate interaction between environmental and genetic variables. The EF-hand proteins, S100A8 and S100A9 act as pro-inflammatory mediators and amyloidogenic proteins. They are implicated in the etiology of several diseases and pivotally influence many cellular functions such as danger signaling and motility. This study investigates the pathogenic roles of S100A8 and S100A9 in the PD model. The recombinant S100A8, S100A9, and SFPQ proteins (splicing factor proline and glutamine-rich) were purified using conventional techniques.
The consequences of S100A8 and S100A9 treatment, with or without 1-methyl-4-phenylpyridinium (MPP+), were evaluated by MTT assay, annexin V-FITC apoptosis staining, Western blot, and immunocytochemistry analysis. Interaction of S100A8 and S100A9 with SFPQ was identified by conducting pull-down assay and surface plasmon resonance (SPR). The prevalence of S100A8, S100A9, and SFPQ in normal and PD brain tissues was investigated by using Immunofluorescence and Western blot. The results uncovered the apoptotic roles of S100A8 and S100A9 in MPP+-induced SH- SY5Y cells. S100A8 and S100A9 impaired cell apoptosis by regulating JNK and p38-MAPK, increasing the expression of apoptosis-associated proteins, and caspase 9/3 in MPP+-induced SH-SY5Y cells. Besides, the combo also enhanced the expression of the proteins associated with PD, namely a- synuclein (a-syn), PINK1, and Parkin. Moreover, this study identified SFPQ as a functional interacting partner of S100A8 and S100A9 in PD pathophysiology. S100A8 and S100A9 were shown to directly interact with SFPQ with an apparent dissociation constant of 25.0 ± 0.1 nM and 20 ± 0.2 nM, respectively. Interestingly, S100A8 and S100A9 treatment promoted the cytoplasmic mislocalization of SFPQ, most likely impairing the nuclear regulatory functions of SPFQ. The siRNA experiments confirmed that SFPQ is not only interacting with S100A8 and S100A9 but also intricate in apoptosis of MPP+-induced SH-SY5Y cells, employing the same pathway. In line with the findings from SH-SY5Y cells, the primary neuron cells from rat brain validated that S100A8 aggravates apoptosis. S100A8 + MPP+ treated primary neuron cells showed marked dislocation of SFPQ to the cytoplasm and colocalization with S100A8. In the postmortem brain of PD patients, S100A8 and S100A9 were significantly upregulated compared with control samples, emphasizing their pathological consequences. In a nutshell, the findings from this study highlighted the apoptotic roles of S100A8 and S100A9 in association with SFPQ, as a critical pathway contributing to the progression of PD.

목차 (Table of Contents)

• Ⅰ. INTRODUCTION = 1
• Ⅱ. MATERIALS AND METHODS = 7
• 2.1. Reagents and antibodies = 7
• 2.2. Production of recombinant proteins = 8
• 2.3. Cell culture = 11
• 2.4. Cell viability assay = 12
• 2.5. Apoptosis detection = 12
• 2.6. Western blot = 13
• 2.7. Nickle affinity pull-down assay = 14
• 2.8. Surface plasmon resonance = 14
• 2.9. Immunocytochemistry = 15
• 2.10. Small interfering RNA (siRNA) transfection = 16
• 2.11. Prediction and modeling of S100A8 and S100A9 binding site on SFPQ = 17
• 2.12. Human tissue samples = 18
• 2.13. Immunofluorescence = 18
• 2.14. Statistical analysis = 19
• Ⅲ. RESULTS = 20
• 3.1. The pathogenic role of S100A8 and S100A9 = 20
• 3.1.1. Cloning and purification of recombinant S100A8 and S100A9 = 20
• 3.1.2. Effects of S100A8 and S100A9 on MPP+-induced cytotoxicity = 23
• 3.2. Pathogenic mechanism of S100A8 and S100A9 = 29
• 3.2.1. Effects of S100A8 and S100A9 on MAPK signaling proteins = 29
• 3.2.2. Effects of S100A8 and S100A9 on PD-associated proteins = 38
• 3.3. Identification of SFPQ as S100A8 and S100A9 interacting protein = 41
• 3.3.1. Cloning and purification of recombinant SFPQ = 45
• 3.3.2. Interaction of S100A8 and S100A9 with recombinant SFPQ = 49
• 3.4. S100A8 and S100A9 prevalence in primary neuron cells and human PD patients = 82
• 3.4.1. S100A8, S100A9 and their interaction with SFPQ in primary neuron cells = 82
• 3.4.2. S100A8, S100A9 and SFPQ in PD patient = 83
• Ⅳ. DISCUSSION = 89
• Ⅴ. CONCLUSION = 93
• REFERENCES = 94
• 국문요지 = 111

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