Title Page
Contents
General Overview 9
1. Mitochondrial Quality Control in Neurodegenerative Diseases 9
2. Role of Cellular Prion Protein in Various Cancer 10
Chapter 1. Impairment of Neuronal Mitochondrial Quality Control in Prion-Induced Neurodegeneration 13
I. Introduction 14
II. Materials and Methods 17
1. Human and Animal Models 17
2. Primary Cortical Neuronal (CxN) Cell Cultures and Scrapie Infection 19
3. Western Blot Analysis 19
4. Subcellular Fractionation 20
5. Immunohistochemistry and Immunofluorescence 20
6. Image Acquisition and Quantification 21
7. Cell Viability Assay 21
8. DNA Construction 22
9. Mitophagy Assay 22
10. Measurement of Mitochondrial Membrane Potential (△Ψm) and mtROS 22
11. Determination of Glycolysis and ATP Levels 23
12. Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR) 23
13. Measurement of Mitochondrial Length 24
14. Caspase Activity Assay 25
15. Statistical Analysis 25
III. Results 26
1. Scrapie Infection Induces Mitochondrial ROS (mtROS) Production and Reduces Mitochondrial Membrane Potential (△Ψm) 26
2. Scrapie Infection Regulates Drp1-mediated Mitochondrial Fission 29
3. Scrapie Infection Promotes Mitophagy 35
4. Scrapie Infection Impairs Mitochondrial Oxidative Phosphorylation (OXPHOS) and Alters Glycolytic Metabolism 39
5. Scrapie Infection Induces Apoptosis through Mitochondrial Dysfunction 41
6. Mitochondrial Quality Control Is Impaired in sCJD Brains 43
IV. Discussion 46
Chapter 2. Cellular Prion Protein Is Closely Associated with Early Recurrence and Poor Survival in Patients with Hepatocellular Carcinoma 50
I. Introduction 51
II. Materials and Methods 53
1. Human Patients and Tissue Microarray 53
2. Immunohistochemical Staining of Cellular Prion Protein in Human HCC Tissue Microarray 53
3. Evaluation of Immunostaining 54
4. Statistical Analysis 54
III. Results 56
1. Baseline Characteristics of Enrolled Patients 56
2. Differences According to PrPC Expression[이미지참조] 58
3. Association of PrPC Expression with Survival of Patients with HCC[이미지참조] 60
4. Univariate and Multivariate Analysis of Overall Survival 62
5. Association of PrPC Expression with Recurrence in Patients with HCC[이미지참조] 65
6. Univariate and Multivariate Analysis of 1-Year Recurrence Following Surgical Reaction 67
IV. Discussion 70
References 74
Abstract in Korean 84
Abstract in English 87
Chapter 1 6
Table 1. Prion diseases in human and animal 14
Table 2. Characterization of human subjects. Control specimens were obtained from individuals without CJD and AD. 18
Table 3. Primers for qRT-PCR 24
Chapter 2 6
Table 1. Clinical characteristics according to PrPC expression[이미지참조] 57
Table 2. Univariate and multivariate analysis of 1-year recurrence following surgical reaction. 63
Table 3. Univariate and multivariate logistic analysis for 1-year recurrence following surgical resection 68
General Overview 7
Figure 1. Mitochondrial quality control mechanisms in various mitochondrial stress response. 9
Figure 2. Function of prion protein (PrPC) in cancer cells.[이미지참조] 11
Chapter 1 7
Figure 1. Scrapie infection induces mitochondrial ROS (mtROS) production and the loss of the mitochondrial membrane potential (△Ψm) followed by mitochondrial fragmentation. 27
Figure 2. Scrapie infection induces phosphorylation of Drp1 at Ser616 (P-Drp1), which colocalizes with PrPSc in the mitochondria.[이미지참조] 30
Figure 3. Scrapie infection induces Drp1-mediated mitochondrial fission in the brains of 22L scrapie-infected mice. 32
Figure 4. RhoA/ROCK activity is involved in Drp1-mediated mitochondrial fission in 22L scrapie-infected CxN cells. The control (CON) or 22L scrapie-infected (22L) CxN cells were... 33
Figure 5. RhoA/ROCK activity is involved in mtROS generation and △Ψm in 22L scrapie-infected CxN cells. The control (CON) or 22L scrapie-infected (22L) CxN cells were... 34
Figure 6. Scrapie infection induces autophagy in mitochondria containing PrPSc.[이미지참조] 37
Figure 7. Scrapie infection induced mitophagy in the brains of 22L scrapie-infected mice. 38
Figure 8. Scrapie infection induces mitochondrial dysfunction associated with defects in OXPHOS-mediated glycolytic metabolism. 40
Figure 9. Scrapie infection induces cell apoptosis by promoting caspase 3 activation. 42
Figure 10. Prion disease induces neuropathogenesis via mitochondrial quality control dysfunction. 45
Figure 11. Schematic representation of PrPSc-mediated neuronal cell death through mitochondrial dysfunction.[이미지참조] 46
Chapter 2 8
Figure 1. PrPC was immunohistochemically stained, and its intensity was graded as 0 (A), 1 (B), 2 (C), and 3 (D).[이미지참조] 59
Figure 2. The cumulative survival rates in HCC patients with high PrPC were significantly lower than those of patients with low PrPC(p=0.043).[이미지참조] 61
Figure 3. Kaplan-Meier curve to compare the cumulative survival rate according to four combinations between tumor size and PrPC expression.[이미지참조] 64
Figure 4. The cumulative recurrence rates in HCC patients with high PrPC were higher than those of patients with low PrPC.[이미지참조] 66
Figure 5. Kaplan-Meier curve to compare the cumulative recurrence rate according to four combinations between tumor size and PrPC expression.[이미지참조] 69