放射線治療在腫瘤治療上是一種十分常見的治療方式，主要是藉由傷害DNA的結構的方式，使得快速分裂增生的細胞死亡而達到腫瘤治療的目的。在臨床放射線治療中，會根據目標腫瘤組織的不同，劑量的選擇上會從1.8 Gy到20 Gy之間來做選擇，而在一般的情況下，2 Gy的劑量即可使得腫瘤細胞死亡。但在放射線治療的過程中，尤其是在腦部的腫瘤治療上，可能會由於放射線的治療，而影響腦部神經的發育，特別是與學習記憶最有關係的海馬迴神經細胞，另外，也有研究指出，在低劑量的放射線治療之下，會增加細胞的存活率和DNA修復，在我們的實驗中已經證實，利用MTT assays發現在低劑量(0.2 Gy)的放射線處理之下會增加細胞的活性，但不會增加細胞數，因此我們認為放射線或許會影響粒線體的活性，在實驗結果中可以明顯的發現，在0.2 Gy 的放射線處理之下，會使得粒線體融合並增長，以及Drp1的蛋白質表現量下降，證實粒線體會走向融合的路徑，但低劑量的放射線並不會增加粒線體的膜電位、氧化程度以及粒線體DNA的複製數目。最後，在我們研究中想了解放射線是否會對受損的神經達到治療的效果，阿茲海默症是常見的失智症之一，在阿茲海默症的病人中常可觀察到大腦中海馬迴受到損害，導致病人的學習記憶能力受損，因此我們使用beta-amyloid (Aβ)處理海馬迴神經細胞，模擬阿茲海默症中受到傷害的神經細胞，並且照射不同劑量的放射線，但在實驗結果中尚未發現放射線有治療的效果。

Irradiation is a common treatment in tumor therapy. Radiation can cause DNA damage and death of proliferating cells. During clinical radiation therapy, the radiation dose could range from 1.8 to 20 Gy depending on targets. In the normal cases, 2 Gy-radiation treatment will cause cancer cell death. In the brain tissue, hippocampal neuron is not as sensitive to radiation. Some studies indicated the low dose radiation would induce cellular protective mechanism to promote cell survival and DNA repair. In our study, we demonstrate the low dose radiation would promote survival of hippocampal neurons based on MTT assays. 0.2 Gy radiation treatment had no effect on the cell number. We hypothesized to investigate the relationship between irradiation and mitochondrial function. However, radiation treatment did not affect membrane potential, ROS level and mitochondrial DNA copy number. Interestingly, the low dose radiation (0.2 Gy) may promote the mitochondria fusion found by immunofluorescence staining. The relative level of Drp1, a fission protein, was decreased after radiation treatment. Hippocampus is the region of the brain that suffers damage in Alzheimer's disease. To determine whether low dosage radiation would have beneficial effect on neurodegenerative disease, we treated hippocampal neuron with beta-amyloid (Aβ) to mimic Alzheimer's pathology followed by radiation therapy. Our results so far did not show protective effect of radiation on Aβ-treated hippocampal neurons.

Abstract I
摘要 II
誌謝 III
Index VI
Abbreviation IX
Introduction 1
Radiation therapy 1
Hippocampal neurons 2
Mitochondrial function 3
Mitochondrial oxidative stress 4
Mitochondrial fusion and fission 5
Mitochondria in neurons 6
Mitochondrial disease 7
Alzheimer's disease 7
Autophagy and Mitophagy 8
Materials and Methods 9
Reagents 9
Primary culture of hippocampal neurons 10
Radiation therapy via linear accelerator 10
MTT assays 11
Cell number counting 11
Immunofluorescence and confocal microscopy 11
Immunoblotting 12
Measurement of mitochondrial membrane potential using Flow cytometry 12
Measurement of mitochondrial ROS level using Flow cytometry 13
Apoptosis analysis using Flow cytometry 13
Genomic DNA extraction 13
Measurement of mitochondrial DNA copy number using real-time polymerase chain reaction (real-time PCR) 14
β-amyloid (25-35) incubation and aggregation 14
Results 15
Low dose radiation increased the level of MTT assays but not numbers of hippocampal neurons. 15
0.2 Gy radiation treatment had no effects on mitochondrial membrane potential, ROS level, and mitochondrial DNA copy number 15
0.2 Gy radiation treatment promoted mitochondrial fusion 17
PSD95 were decreased after 0.2 Gy radiation treatment 18
Radiation treatment had no effect on Aβ-treated hippocampal neurons 18
Discussion 20
References 23
Figures 30
Figure 1. The level of MTT assays in 0.2 Gy radiation-treated neurons was increased compared to control cells. 31
Figure 2. Radiation treatment did not have effects on mitochondrial membrane potential, ROS level, mitochondrial DNA copy number, and proteins of OXPHOS. 33
Figure 3. 0.2 Gy radiation treatment promoted mitochondrial fusion. 35
Figure 4. PSD95 expression levels were decreased in radiation treatment. 36
Figure 5. Radiation treatment did not affect on Aβ-treated hippocampal neurons. 40
Table 41
Table 1. Primary antibody 41

1. Riley, P. A. (1994) Free radicals in biology: oxidative stress and the effects of ionizing radiation. International journal of radiation biology 65, 27-33
2. Walker, M. D., Strike, T. A., and Sheline, G. E. (1979) An analysis of dose-effect relationship in the radiotherapy of malignant gliomas. International journal of radiation oncology, biology, physics 5, 1725-1731
3. Monje, M. L., Mizumatsu, S., Fike, J. R., and Palmer, T. D. (2002) Irradiation induces neural precursor-cell dysfunction. Nature medicine 8, 955-962
4. Posner, J. B. (1992) Management of brain metastases. Revue neurologique 148, 477-487
5. Roman, D. D., and Sperduto, P. W. (1995) Neuropsychological effects of cranial radiation: current knowledge and future directions. International journal of radiation oncology, biology, physics 31, 983-998
6. Abayomi, O. K. (2002) Pathogenesis of cognitive decline following therapeutic irradiation for head and neck tumors. Acta oncologica 41, 346-351
7. Madsen, T. M., Kristjansen, P. E., Bolwig, T. G., and Wortwein, G. (2003) Arrested neuronal proliferation and impaired hippocampal function following fractionated brain irradiation in the adult rat. Neuroscience 119, 635-642
8. Mizumatsu, S., Monje, M. L., Morhardt, D. R., Rola, R., Palmer, T. D., and Fike, J. R. (2003) Extreme sensitivity of adult neurogenesis to low doses of X-irradiation. Cancer research 63, 4021-4027
9. Rola, R., Raber, J., Rizk, A., Otsuka, S., VandenBerg, S. R., Morhardt, D. R., and Fike, J. R. (2004) Radiation-induced impairment of hippocampal neurogenesis is associated with cognitive deficits in young mice. Experimental neurology 188, 316-330
10. Fike, J. R., Rola, R., and Limoli, C. L. (2007) Radiation response of neural precursor cells. Neurosurgery clinics of North America 18, 115-127, x
11. Tan, Y. F., Rosenzweig, S., Jaffray, D., and Wojtowicz, J. M. (2011) Depletion of new neurons by image guided irradiation. Frontiers in neuroscience 5, 59
12. Milner, B. (1972) Disorders of learning and memory after temporal lobe lesions in man. Clinical neurosurgery 19, 421-446
13. Scoville, W. B., and Milner, B. (1957) Loss of recent memory after bilateral hippocampal lesions. Journal of neurology, neurosurgery, and psychiatry 20, 11-21
14. Squire, L. R. (1986) Mechanisms of memory. Science 232, 1612-1619
15. Bird, C. M., and Burgess, N. (2008) The hippocampus and memory: insights from spatial processing. Nature reviews. Neuroscience 9, 182-194
16. Braak, H., Braak, E., and Bohl, J. (1993) Staging of Alzheimer-related cortical destruction. European neurology 33, 403-408
17. Arimura, N., and Kaibuchi, K. (2007) Neuronal polarity: from extracellular signals to intracellular mechanisms. Nature reviews. Neuroscience 8, 194-205
18. Wallace, D. C. (2001) A mitochondrial paradigm for degenerative diseases and ageing. Novartis Foundation symposium 235, 247-263; discussion 263-246
19. Galloway, C. A., and Yoon, Y. (2012) Perspectives on: SGP symposium on mitochondrial physiology and medicine: what comes first, misshape or dysfunction? The view from metabolic excess. The Journal of general physiology 139, 455-463
20. Reichert, A. S., and Neupert, W. (2004) Mitochondriomics or what makes us breathe. Trends in genetics : TIG 20, 555-562
21. Fernie, A. R., Carrari, F., and Sweetlove, L. J. (2004) Respiratory metabolism: glycolysis, the TCA cycle and mitochondrial electron transport. Current opinion in plant biology 7, 254-261
22. Stock, D., Leslie, A. G., and Walker, J. E. (1999) Molecular architecture of the rotary motor in ATP synthase. Science 286, 1700-1705
23. Okuno, D., Iino, R., and Noji, H. (2011) Rotation and structure of FoF1-ATP synthase. Journal of biochemistry 149, 655-664
24. Murphy, M. P. (2009) How mitochondria produce reactive oxygen species. The Biochemical journal 417, 1-13
25. Orrenius, S., Gogvadze, V., and Zhivotovsky, B. (2007) Mitochondrial oxidative stress: implications for cell death. Annual review of pharmacology and toxicology 47, 143-183
26. Koopman, W. J., Nijtmans, L. G., Dieteren, C. E., Roestenberg, P., Valsecchi, F., Smeitink, J. A., and Willems, P. H. (2010) Mammalian mitochondrial complex I: biogenesis, regulation, and reactive oxygen species generation. Antioxidants & redox signaling 12, 1431-1470
27. Han, D., Antunes, F., Canali, R., Rettori, D., and Cadenas, E. (2003) Voltage-dependent anion channels control the release of the superoxide anion from mitochondria to cytosol. The Journal of biological chemistry 278, 5557-5563
28. Zelko, I. N., Mariani, T. J., and Folz, R. J. (2002) Superoxide dismutase multigene family: a comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures, evolution, and expression. Free radical biology & medicine 33, 337-349
29. Mills, G. C. (1957) Hemoglobin catabolism. I. Glutathione peroxidase, an erythrocyte enzyme which protects hemoglobin from oxidative breakdown. The Journal of biological chemistry 229, 189-197
30. Waris, G., and Ahsan, H. (2006) Reactive oxygen species: role in the development of cancer and various chronic conditions. Journal of carcinogenesis 5, 14
31. Uttara, B., Singh, A. V., Zamboni, P., and Mahajan, R. T. (2009) Oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic options. Current neuropharmacology 7, 65-74
32. Liochev, S. I. (2013) Reactive oxygen species and the free radical theory of aging. Free radical biology & medicine 60, 1-4
33. Yakes, F. M., and Van Houten, B. (1997) Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress. Proceedings of the National Academy of Sciences of the United States of America 94, 514-519
34. Chen, H., Vermulst, M., Wang, Y. E., Chomyn, A., Prolla, T. A., McCaffery, J. M., and Chan, D. C. (2010) Mitochondrial fusion is required for mtDNA stability in skeletal muscle and tolerance of mtDNA mutations. Cell 141, 280-289
35. Ono, T., Isobe, K., Nakada, K., and Hayashi, J. I. (2001) Human cells are protected from mitochondrial dysfunction by complementation of DNA products in fused mitochondria. Nature genetics 28, 272-275
36. Fritz, S., Rapaport, D., Klanner, E., Neupert, W., and Westermann, B. (2001) Connection of the mitochondrial outer and inner membranes by Fzo1 is critical for organellar fusion. The Journal of cell biology 152, 683-692
37. Rojo, M., Legros, F., Chateau, D., and Lombes, A. (2002) Membrane topology and mitochondrial targeting of mitofusins, ubiquitous mammalian homologs of the transmembrane GTPase Fzo. Journal of cell science 115, 1663-1674
38. Santel, A., and Fuller, M. T. (2001) Control of mitochondrial morphology by a human mitofusin. Journal of cell science 114, 867-874
39. Cipolat, S., Martins de Brito, O., Dal Zilio, B., and Scorrano, L. (2004) OPA1 requires mitofusin 1 to promote mitochondrial fusion. Proceedings of the National Academy of Sciences of the United States of America 101, 15927-15932
40. Smirnova, E., Shurland, D. L., Ryazantsev, S. N., and van der Bliek, A. M. (1998) A human dynamin-related protein controls the distribution of mitochondria. The Journal of cell biology 143, 351-358
41. Zhang, Y., and Chan, D. C. (2007) Structural basis for recruitment of mitochondrial fission complexes by Fis1. Proceedings of the National Academy of Sciences of the United States of America 104, 18526-18530
42. Otera, H., Wang, C., Cleland, M. M., Setoguchi, K., Yokota, S., Youle, R. J., and Mihara, K. (2010) Mff is an essential factor for mitochondrial recruitment of Drp1 during mitochondrial fission in mammalian cells. The Journal of cell biology 191, 1141-1158
43. Nicholls, D. G., and Budd, S. L. (2000) Mitochondria and neuronal survival. Physiological reviews 80, 315-360
44. Sheng, Z. H., and Cai, Q. (2012) Mitochondrial transport in neurons: impact on synaptic homeostasis and neurodegeneration. Nature reviews. Neuroscience 13, 77-93
45. Kang, J. S., Tian, J. H., Pan, P. Y., Zald, P., Li, C., Deng, C., and Sheng, Z. H. (2008) Docking of axonal mitochondria by syntaphilin controls their mobility and affects short-term facilitation. Cell 132, 137-148
46. Hollenbeck, P. J., and Saxton, W. M. (2005) The axonal transport of mitochondria. Journal of cell science 118, 5411-5419
47. Tanaka, Y., Kanai, Y., Okada, Y., Nonaka, S., Takeda, S., Harada, A., and Hirokawa, N. (1998) Targeted disruption of mouse conventional kinesin heavy chain, kif5B, results in abnormal perinuclear clustering of mitochondria. Cell 93, 1147-1158
48. Pilling, A. D., Horiuchi, D., Lively, C. M., and Saxton, W. M. (2006) Kinesin-1 and Dynein are the primary motors for fast transport of mitochondria in Drosophila motor axons. Molecular biology of the cell 17, 2057-2068
49. Cai, Q., Gerwin, C., and Sheng, Z. H. (2005) Syntabulin-mediated anterograde transport of mitochondria along neuronal processes. The Journal of cell biology 170, 959-969
50. Kanai, Y., Okada, Y., Tanaka, Y., Harada, A., Terada, S., and Hirokawa, N. (2000) KIF5C, a novel neuronal kinesin enriched in motor neurons. The Journal of neuroscience : the official journal of the Society for Neuroscience 20, 6374-6384
51. Burbulla, L. F., Krebiehl, G., and Kruger, R. (2010) Balance is the challenge--the impact of mitochondrial dynamics in Parkinson's disease. European journal of clinical investigation 40, 1048-1060
52. Selkoe, D. J. (2001) Alzheimer's disease: genes, proteins, and therapy. Physiological reviews 81, 741-766
53. Druzhyna, N. M., Wilson, G. L., and LeDoux, S. P. (2008) Mitochondrial DNA repair in aging and disease. Mechanisms of ageing and development 129, 383-390
54. Taylor, R. W., and Turnbull, D. M. (2005) Mitochondrial DNA mutations in human disease. Nature reviews. Genetics 6, 389-402
55. Detmer, S. A., Vande Velde, C., Cleveland, D. W., and Chan, D. C. (2008) Hindlimb gait defects due to motor axon loss and reduced distal muscles in a transgenic mouse model of Charcot-Marie-Tooth type 2A. Human molecular genetics 17, 367-375
56. Huang, H. C., and Jiang, Z. F. (2009) Accumulated amyloid-beta peptide and hyperphosphorylated tau protein: relationship and links in Alzheimer's disease. Journal of Alzheimer's disease : JAD 16, 15-27
57. Yin, Y. I., Bassit, B., Zhu, L., Yang, X., Wang, C., and Li, Y. M. (2007) {gamma}-Secretase Substrate Concentration Modulates the Abeta42/Abeta40 Ratio: IMPLICATIONS FOR ALZHEIMER DISEASE. The Journal of biological chemistry 282, 23639-23644
58. Schmidt, M., Sachse, C., Richter, W., Xu, C., Fandrich, M., and Grigorieff, N. (2009) Comparison of Alzheimer Abeta(1-40) and Abeta(1-42) amyloid fibrils reveals similar protofilament structures. Proceedings of the National Academy of Sciences of the United States of America 106, 19813-19818
59. Song, J., Park, K. A., Lee, W. T., and Lee, J. E. (2014) Apoptosis signal regulating kinase 1 (ASK1): potential as a therapeutic target for Alzheimer's disease. International journal of molecular sciences 15, 2119-2129
60. Mines, M. A., Beurel, E., and Jope, R. S. (2011) Regulation of cell survival mechanisms in Alzheimer's disease by glycogen synthase kinase-3. International journal of Alzheimer's disease 2011, 861072
61. Rui, Y., Gu, J., Yu, K., Hartzell, H. C., and Zheng, J. Q. (2010) Inhibition of AMPA receptor trafficking at hippocampal synapses by beta-amyloid oligomers: the mitochondrial contribution. Molecular brain 3, 10
62. Levine, B., and Klionsky, D. J. (2004) Development by self-digestion: molecular mechanisms and biological functions of autophagy. Developmental cell 6, 463-477
63. Deretic, V., Saitoh, T., and Akira, S. (2013) Autophagy in infection, inflammation and immunity. Nature reviews. Immunology 13, 722-737
64. Hayashi-Nishino, M., Fujita, N., Noda, T., Yamaguchi, A., Yoshimori, T., and Yamamoto, A. (2009) A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation. Nature cell biology 11, 1433-1437
65. Yla-Anttila, P., Vihinen, H., Jokitalo, E., and Eskelinen, E. L. (2009) 3D tomography reveals connections between the phagophore and endoplasmic reticulum. Autophagy 5, 1180-1185
66. Mizushima, N. (2010) The role of the Atg1/ULK1 complex in autophagy regulation. Current opinion in cell biology 22, 132-139
67. Itakura, E., and Mizushima, N. (2010) Characterization of autophagosome formation site by a hierarchical analysis of mammalian Atg proteins. Autophagy 6, 764-776
68. Nakatogawa, H., Suzuki, K., Kamada, Y., and Ohsumi, Y. (2009) Dynamics and diversity in autophagy mechanisms: lessons from yeast. Nature reviews. Molecular cell biology 10, 458-467
69. Weidberg, H., Shvets, E., Shpilka, T., Shimron, F., Shinder, V., and Elazar, Z. (2010) LC3 and GATE-16/GABARAP subfamilies are both essential yet act differently in autophagosome biogenesis. The EMBO journal 29, 1792-1802
70. Youle, R. J., and Narendra, D. P. (2011) Mechanisms of mitophagy. Nature reviews. Molecular cell biology 12, 9-14
71. Youle, R. J., and van der Bliek, A. M. (2012) Mitochondrial fission, fusion, and stress. Science 337, 1062-1065
72. Ashrafi, G., and Schwarz, T. L. (2013) The pathways of mitophagy for quality control and clearance of mitochondria. Cell death and differentiation 20, 31-42
73. Twig, G., Hyde, B., and Shirihai, O. S. (2008) Mitochondrial fusion, fission and autophagy as a quality control axis: the bioenergetic view. Biochimica et biophysica acta 1777, 1092-1097
74. Dagda, R. K., Cherra, S. J., 3rd, Kulich, S. M., Tandon, A., Park, D., and Chu, C. T. (2009) Loss of PINK1 function promotes mitophagy through effects on oxidative stress and mitochondrial fission. The Journal of biological chemistry 284, 13843-13855
75. Tanaka, A., Cleland, M. M., Xu, S., Narendra, D. P., Suen, D. F., Karbowski, M., and Youle, R. J. (2010) Proteasome and p97 mediate mitophagy and degradation of mitofusins induced by Parkin. The Journal of cell biology 191, 1367-1380
76. Monje, M. (2008) Cranial radiation therapy and damage to hippocampal neurogenesis. Developmental disabilities research reviews 14, 238-242
77. Park, H. S., Seong, K. M., Kim, J. Y., Kim, C. S., Yang, K. H., Jin, Y. W., and Nam, S. Y. (2013) Chronic low-dose radiation inhibits the cells death by cytotoxic high-dose radiation increasing the level of AKT and acinus proteins via NF-kappaB activation. International journal of radiation biology 89, 371-377
78. Luckey, T. D. (2006) Radiation hormesis: the good, the bad, and the ugly. Dose-response : a publication of International Hormesis Society 4, 169-190
79. Pollycove, M., and Feinendegen, L. E. (2003) Radiation-induced versus endogenous DNA damage: possible effect of inducible protective responses in mitigating endogenous damage. Human & experimental toxicology 22, 290-306; discussion 307, 315-297, 319-223
80. Wei, L. C., Ding, Y. X., Liu, Y. H., Duan, L., Bai, Y., Shi, M., and Chen, L. W. (2012) Low-dose radiation stimulates Wnt/beta-catenin signaling, neural stem cell proliferation and neurogenesis of the mouse hippocampus in vitro and in vivo. Current Alzheimer research 9, 278-289