臺灣博碩士論文加值系統

帕金森氏症(Parkinson’s disease)是常見的一種神經退化性疾病。帕金森氏症病患的病症有顫抖、身體僵直、運動遲緩，並且無法自主行動，主要的病理特徵是位於人類中腦黑質緻密區中多巴胺神經元有逐漸退化死亡的情形。過去研究顯示氧化壓力的形成與多巴胺神經元的缺失具有相關性。多巴胺為一種神經毒會造成腦內氧化壓力的增加。另外，在帕金森氏症病患的病理組織切片發現位於黑質緻密區的鋅離子含量有明顯增加的情形。實驗室先前的研究顯示出鋅離子與多巴胺造成的神經細胞死亡具有協同作用，並且在大鼠腦部紋狀體區域發現有多巴胺減少的現象，但相關的分子機制仍然未知。因此在本篇研究，我們建立了一個具有鋅離子與多巴胺協同作用導致神經元死亡的小鼠模式。在我們所建立的小鼠模式發現，分別在黑質緻密區注入鋅離子；紋狀體區域注入多巴胺並不會導致多巴胺嚴重受損的情形。然而，同時在黑質緻密區注入鋅離子與紋狀體區域注入多巴胺，可以觀察到紋狀體區域的多巴胺有嚴重受損的情形，並且具有協同作用。為了進一步探討氧化壓力的形成，我們在建立的小鼠模式中處理抗氧化劑乙醯硫胱胺酸(N-acetylcysteine, NAC)或榖胱甘肽(glutathione, GSH)，再分別在黑質緻密區注入鋅離子與紋狀體注入多巴胺去看是否具有保護的作用。結果顯示乙醯硫胱胺酸對於鋅離子與多巴胺造成多巴胺神經元死亡的現象具有保護作用。這些結果顯示鋅離子與多巴胺可能經由自體吞噬作用(autophagy)與內質網調控細胞死亡(endoplasmic reticulum; ER)的分子調控路徑造成多巴胺神經元死亡。

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases. The clinical symptoms of PD are tremor, rigidity, bradykinesia, and autonomic dysfunctions. The pathological feature of PD is the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Extensive studies have shown the involvement of oxidative stress in the loss of dopaminergic neurons. Dopamine (DA) is a possible neurotoxin to increase oxidative stress. Furthermore, postmortem studies show that the Zn2+ level is increased in SNpc. Our previous study revealed that Zn2+ and DA synergistically caused neuronal cell death and DA depletion in the rat striatum, but the molecular mechanisms remain unclear. In this study, we established a mouse model for the study of synergistic effects of Zn2+ and DA. The result showed that individual infusion of Zn2+ and DA in SN and striatum, respectively, did not cause severe damages. However, simultaneous infusion of Zn2+ and DA synergistically elicited a significant depletion of the striatal DA content. Furthermore, to determine the antioxidant effect on the Zn2+- and DA- induced degeneration of dopaminergic neurons, various concentrations of N-acetylcysteine (NAC)- or glutathione (GSH) were infused with Zn2+ and DA into SN or striatum. We found that NAC but not GSH significantly rescued the death of dopaminergic neurons induced by Zn2+ and DA. Our results suggest that Zn2+- and DA- induced degeneration of mouse dopaminergic neurons may be mediated by the activation of autophagy and ER stress.

Abstract 1
摘要 2
Abbreviations 3
Chapter 1: Introduction 5
1.1. Parkinson’s disease (PD) 5
1.2. Nigrostriatal dopaminergic pathway 5
1.3. Dopamine (DA) 6
1.3.1 Characterization of DA 6
1.3.2 Neurotoxicity of DA 7
1.4. Zinc 7
1.4.1. Characterization of Zinc 7
1.4.2. Neurotoxicity of Zinc 8
1.5. Molecular pathways of nigral degeneration 8
1.5.1. Oxidative stress 8
1.5.2. Endoplasmic reticulum stress 9
1.5.3. Apoptosis 10
1.5.4. Autophagy 10
1.5.5. Inflammation 11
1.5.6. JAK-STAT pathway 12
1.6. Specific Aims 13
Chapter 2: Materials and Method 14
2.1. Animals 14
2.2. Stereotaxic surgery 14
2.3. Animal perfusion and fixation 15
2.4. Tissue frozen section 15
2.5. Immunohistochemistry (IHC) 15
2.6. Immunofluorescent staining (IF) 16
2.7. Cell Counting and Optical Densitometry Analysis 17
2.8. HPLC-EC analysis 17
2.9. Western blotting 18
2.10. Statistics 19
Chapter 3: Results 20
3.1. The synergistic effects of Zn2+- and DA-induced striatal DA depletion in mouse model 20
3.2. Characterization of the synergistic effects of Zn2+ and DA on ST and SN. 21
3.2.1 Zn2+- and DA-induced neurotoxicity in striatal DA and DA metabolites content by HPLC-EC 21
3.2.2 Zn2+- and DA-induced neurotoxicity on the number of TH+ neurons
in SNpc. 21
3.3. The antioxidant effect of Zn2+- and DA-induced neurotoxicity on TH+ immunoreactivity. 22
3.3.1 No significant effect of GSH on the Zn2+- and DA-induced
neurotoxicity 22
3.3.2 The protective effect of NAC on the Zn2+- and DA-induced neurotoxicity
22
3.4. The possible signaling pathway involved in the synergistic effects of Zn2+- and DA-induced degeneration of mouse dopaminergic neurons. 24
3.4.1 Zn2+- and DA-induced autophagy in SN and ST. 24
3.4.2 ER pathway may involve in Zn2+- and DA-induced neuronal death in SN
and ST 25
3.4.3 Apoptosis was not detected in Zn2+- and DA-infused SN and ST 25
3.4.4 Inflammation may not involve in Zn2+- and DA-induced degeneration of dopaminergic neurons. 26
3.4.5 The role of JAK-STAT pathway in Zn2+- and DA-induced damages in SN
and ST. . 26
Chapter 4: Discussion 28
Reference 32
Figures 38
Figure 1. Quantification of TH+ fibers density in the ST by optical densitometry analysis 38
Figure 2. No significant damages were detected in one week after infusions of DA and Zn2+ on TH+ immunoreactivity. 42
Figure 3. Synergistic effects of Zn2+ and DA on TH+ immunoreactivity 43
Figure 4. The neurotoxic effects of Zn2+ and DA on the nigrostriatal dopaminergic system. 45
Figure 5. Effects of infusions of Zn2+ and DA on the number of TH+ neurons in SNpc. 47
Figure 6. Effects of GSH on Zn2+- and DA-induced neurotoxicity: various concentrations of GSH. 49
Figure 7. Effects of NAC on Zn2+- and DA-induced neurotoxicity: various concentrations of NAC. 51
Figure 8. The antioxidant effect of NAC on Zn2+- and DA-induced neurotoxicity. 53
Figure 9. Infusions of Zn2+ and DA induced activation of autophagy pathway in
SN and ST 54
Figure 10. ER stress was elicited by infusions of Zn2+ and DA evidenced by the activation of caspase 12 56
Figure 11. Infusions of Zn2+ and DA did not trigger mitochondria-dependent apoptosis. 58
Figure 12. No alterations on inflammation pathways were found upon treatments
of Zn2+ and DA in ST and SN. 60
Figure 13. Effects of Zn2+ and DA on the expression STAT1 in ST and SN 63
Figure 14. Effects of Zn2+ and DA on the activation of p-STAT1 (Ser727) 65
Figure 15. Effects of Zn2+ and DA on the expression of STAT3 67
Figure 16. Effects of Zn2+ and DA on the activation of p-STAT3 (Ser727) 69
Appendix1. Hypothetical mechanism of DA toxicity 71
Appendix2. Neuronal death pathways activated by Zn2+ 72

Akira, S. (1999). Functional roles of STAT family proteins: lessons from knockout mice. Stem cells (Dayton, Ohio) 17, 138-146.
Anglade, P., Vyas, S., Hirsch, E.C., and Agid, Y. (1997). Apoptosis in dopaminergic neurons of the human substantia nigra during normal aging. Histology and histopathology 12, 603-610.
Beyersmann, D., and Haase, H. (2001). Functions of zinc in signaling, proliferation and differentiation of mammalian cells. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine 14, 331-341.
Blum, D., Torch, S., Lambeng, N., Nissou, M., Benabid, A.L., Sadoul, R., and Verna, J.M. (2001). Molecular pathways involved in the neurotoxicity of 6-OHDA, dopamine and MPTP: contribution to the apoptotic theory in Parkinson's disease. Progress in neurobiology 65, 135-172.
Cadet, J.L., and Brannock, C. (1998). Free radicals and the pathobiology of brain dopamine systems. Neurochemistry international 32, 117-131.
Collier, T.J., Kanaan, N.M., and Kordower, J.H. (2011). Ageing as a primary risk factor for Parkinson's disease: evidence from studies of non-human primates. Nature reviews Neuroscience 12, 359-366.
Cuajungco, M.P., and Lees, G.J. (1997a). Zinc and Alzheimer's disease: is there a direct link? Brain research Brain research reviews 23, 219-236.
Cuajungco, M.P., and Lees, G.J. (1997b). Zinc metabolism in the brain: relevance to human neurodegenerative disorders. Neurobiol Dis 4, 137-169.
Dauer, W., and Przedborski, S. (2003). Parkinson's disease: mechanisms and models. Neuron 39, 889-909.
Denes, A., Lopez-Castejon, G., and Brough, D. (2012). Caspase-1: is IL-1 just the tip of the ICEberg? Cell Death and Disease 3, e338.
Deumens, R., Blokland, A., and Prickaerts, J. (2002). Modeling Parkinson's Disease in Rats: An Evaluation of 6-OHDA Lesions of the Nigrostriatal Pathway. Experimental Neurology 175, 303-317.
Dexter, D.T., Carayon, A., Javoy-Agid, F., Agid, Y., Wells, F.R., Daniel, S.E., Lees, A.J., Jenner, P., and Marsden, C.D. (1991). Alterations in the levels of iron, ferritin and other trace metals in Parkinson's disease and other neurodegenerative diseases affecting the basal ganglia. Brain : a journal of neurology 114 ( Pt 4), 1953-1975.
Dringen, R. (2000). Metabolism and functions of glutathione in brain. Progress in neurobiology 62, 649-671.
Durieux, P.F., Schiffmann, S.N., and de Kerchove d'Exaerde, A. (2011). Differential regulation of motor control and response to dopaminergic drugs by D1R and D2R neurons in distinct dorsal striatum subregions. The EMBO Journal 31, 640-653.
Elmore, S. (2007). Apoptosis: A Review of Programmed Cell Death. Toxicologic Pathology 35, 495-516.
Fahn, S. (2003). Description of Parkinson's disease as a clinical syndrome. Annals of the New York Academy of Sciences 991, 1-14.
Frederickson, C.J., Suh, S.W., Silva, D., Frederickson, C.J., and Thompson, R.B. (2000). Importance of zinc in the central nervous system: the zinc-containing neuron. The Journal of nutrition 130, 1471S-1483S.
Friedman, L.G., Lachenmayer, M.L., Wang, J., He, L., Poulose, S.M., Komatsu, M., Holstein, G.R., and Yue, Z. (2012). Disrupted Autophagy Leads to Dopaminergic Axon and Dendrite Degeneration and Promotes Presynaptic Accumulation of -Synuclein and LRRK2 in the Brain. Journal of Neuroscience 32, 7585-7593.
Gerfen, C.R. (2000). Molecular effects of dopamine on striatal-projection pathways. Trends in neurosciences 23, S64-70.
Giménez-Xavier, P., Francisco, R., Santidrián, A.F., Gil, J., and Ambrosio, S. (2009). Effects of dopamine on LC3-II activation as a marker of autophagy in a neuroblastoma cell model. NeuroToxicology 30, 658-665.
Hartmann, A., Hunot, S., Michel, P.P., Muriel, M.P., Vyas, S., Faucheux, B.A., Mouatt-Prigent, A., Turmel, H., Srinivasan, A., Ruberg, M., et al. (2000). Caspase-3: A vulnerability factor and final effector in apoptotic death of dopaminergic neurons in Parkinson's disease. Proceedings of the National Academy of Sciences 97, 2875-2880.
Inagi, R. (2010). Endoplasmic reticulum stress as a progression factor for kidney injury. Current Opinion in Pharmacology 10, 156-165.
Jenner, P. (2003). Oxidative stress in Parkinson's disease. Annals of Neurology 53, S26-S38.
Kallen, C.J.H., Greevenbroek, M.M.J., Stehouwer, C.D.A., and Schalkwijk, C.G. (2009). Endoplasmic reticulum stress-induced apoptosis in the development of diabetes: is there a role for adipose tissue and liver? Apoptosis 14, 1424-1434.
Kang, R., Zeh, H.J., Lotze, M.T., and Tang, D. (2011). The Beclin 1 network regulates autophagy and apoptosis. Cell Death and Differentiation 18, 571-580.
Kerksick, C., and Willoughby, D. (2005). The antioxidant role of glutathione and N-acetyl-cysteine supplements and exercise-induced oxidative stress. Journal of the International Society of Sports Nutrition 2, 38-44.
Kumar, A., Singh, B.K., Ahmad, I., Shukla, S., Patel, D.K., Srivastava, G., Kumar, V., Pandey, H.P., and Singh, C. (2012). Involvement of NADPH oxidase and glutathione in zinc-induced dopaminergic neurodegeneration in rats: Similarity with paraquat neurotoxicity. Brain Research 1438, 48-64.
Kurokawa, M., and Kornbluth, S. (2009). Caspases and Kinases in a Death Grip. Cell 138, 838-854.
Lee, J.-Y., Son, H.J., Choi, J.H., Cho, E., Kim, J., Chung, S.J., Hwang, O., and Koh, J.-Y. (2009). Cytosolic labile zinc accumulation in degenerating dopaminergic neurons of mouse brain after MPTP treatment. Brain Research 1286, 208-214.
Lichten, L.A., and Cousins, R.J. (2009). Mammalian Zinc Transporters: Nutritional and Physiologic Regulation. Annual Review of Nutrition 29, 153-176.
Lin, A.M.Y., Fan, S.F., Yang, D.M., Hsu, L.L., and Yang, C.H.J. (2003). Zinc-induced apoptosis in substantia nigra of rat brain: neuroprotection by vitamin D3. Free Radical Biology and Medicine 34, 1416-1425.
Lo, H.-S., Chiang, H.-C., Lin, A.M.Y., Chiang, H.-Y., Chu, Y.-C., and Kao, L.-S. (2004). Synergistic effects of dopamine and Zn2+ on the induction of PC12 cell death and dopamine depletion in the striatum: possible implication in the pathogenesis of Parkinson's disease. Neurobiology of Disease 17, 54-61.
Lushchak, V.I. (2012). Glutathione Homeostasis and Functions: Potential Targets for Medical Interventions. Journal of Amino Acids 2012, 1-26.
McLaughlin, B.A., Nelson, D., Erecinska, M., and Chesselet, M.F. (1998). Toxicity of dopamine to striatal neurons in vitro and potentiation of cell death by a mitochondrial inhibitor. J Neurochem 70, 2406-2415.
Mogi, M., Togari, A., Ogawa, M., Ikeguchi, K., Shizuma, N., Fan, D., Nakano, I., and Nagatsu, T. (1998). Effects of repeated systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to mice on interleukin-1beta and nerve growth factor in the striatum. Neuroscience letters 250, 25-28.
Momoi, T. (2004). Caspases involved in ER stress-mediated cell death. Journal of chemical neuroanatomy 28, 101-105.
Novikova, L., Garris, B.L., Garris, D.R., and Lau, Y.S. (2006). Early signs of neuronal apoptosis in the substantia nigra pars compacta of the progressive neurodegenerative mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/probenecid model of Parkinson’s disease. Neuroscience 140, 67-76.
Olanow, C.W. (2007). The pathogenesis of cell death in Parkinson's disease – 2007. Movement Disorders 22, S335-S342.
Rawlings, J.S. (2004). The JAK/STAT signaling pathway. Journal of Cell Science 117, 1281-1283.
Rubinsztein, D.C., DiFiglia, M., Heintz, N., Nixon, R.A., Qin, Z.H., Ravikumar, B., Stefanis, L., and Tolkovsky, A. (2005). Autophagy and its possible roles in nervous system diseases, damage and repair. Autophagy 1, 11-22.
Ryu, E.J., Harding, H.P., Angelastro, J.M., Vitolo, O.V., Ron, D., and Greene, L.A. (2002). Endoplasmic reticulum stress and the unfolded protein response in cellular models of Parkinson's disease. The Journal of neuroscience : the official journal of the Society for Neuroscience 22, 10690-10698.
Sensi, S.L., Paoletti, P., Bush, A.I., and Sekler, I. (2009). Zinc in the physiology and pathology of the CNS. Nature Reviews Neuroscience 10, 780-791.
Shao, W., Zhang, S.-z., Tang, M., Zhang, X.-h., Zhou, Z., Yin, Y.-q., Zhou, Q.-b., Huang, Y.-y., Liu, Y.-j., Wawrousek, E., et al. (2012). Suppression of neuroinflammation by astrocytic dopamine D2 receptors via αB-crystallin. Nature 494, 90-94.
Simon, A.R., Rai, U., Fanburg, B.L., and Cochran, B.H. (1998). Activation of the JAK-STAT pathway by reactive oxygen species. The American journal of physiology 275, C1640-1652.
Stephanou, A., Brar, B.K., Knight, R.A., and Latchman, D.S. (2000). Opposing actions of STAT-1 and STAT-3 on the Bcl-2 and Bcl-x promoters. Cell Death Differ 7, 329-330.
Stoof, J.C., and Kebabian, J.W. (1981). Opposing roles for D-1 and D-2 dopamine receptors in efflux of cyclic AMP from rat neostriatum. Nature 294, 366-368.
Szegezdi, E., Logue, S.E., Gorman, A.M., and Samali, A. (2006). Mediators of endoplasmic reticulum stress-induced apoptosis. EMBO reports 7, 880-885.
Takagi, Y., Harada, J., Chiarugi, A., and Moskowitz, M.A. (2002). STAT1 is activated in neurons after ischemia and contributes to ischemic brain injury. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 22, 1311-1318.
Teismann, P., and Schulz, J.r.B. (2004). Cellular pathology of Parkinson?s disease: astrocytes, microglia and inflammation. Cell and Tissue Research 318, 149-161.
Wood, P.L. (1983). Opioid regulation of CNS dopaminergic pathways: a review of methodology, receptor types, regional variations and species differences. Peptides 4, 595-601.
Xuan, Y.T. (2005). Role of the Protein Kinase C- -Raf-1-MEK-1/2-p44/42 MAPK Signaling Cascade in the Activation of Signal Transducers and Activators of Transcription 1 and 3 and Induction of Cyclooxygenase-2 After Ischemic Preconditioning. Circulation 112, 1971-1978.
Yadav, A. (2005). JAK/STAT3 Pathway Is Involved in Survival of Neurons in Response to Insulin-like Growth Factor and Negatively Regulated by Suppressor of Cytokine Signaling-3. Journal of Biological Chemistry 280, 31830-31840.
Yamashita, T., Sawamoto, K., Suzuki, S., Suzuki, N., Adachi, K., Kawase, T., Mihara, M., Ohsugi, Y., Abe, K., and Okano, H. (2005). Blockade of interleukin-6 signaling aggravates ischemic cerebral damage in mice: possible involvement of Stat3 activation in the protection of neurons. Journal of Neurochemistry 94, 459-468.