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研究生:林之琳
研究生(外文):Zhin-Lin Lin
論文名稱:SonicHedgehog對於三硝基丙酸之神經保護作用及其機制
論文名稱(外文):Neuroprotective effects of Sonic hedgehog against mitochondrial dysfunction in cultured rat cortical neurons
指導教授:楊定一楊定一引用關係
指導教授(外文):Ding-I Yang
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:腦科學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
畢業學年度:97
語文別:英文
論文頁數:75
中文關鍵詞:音速小子三硝基丙酸但丁頓舞蹈症神經保護機制粒線體抑制劑神經細胞
外文關鍵詞:Sonic Hedgehog3-Nitropropionic acidHuntington’s diseaseneuroprotective effectcGMP-dependent protein kinasenuclear factor-kappaB
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背景:Sonic Hedgehog(以下簡稱SHH)是一種在脊椎動物身上調控胚胎時期神經系統發育與分化的蛋白質,而最近有報導指出SHH對於某些神經細胞可能產生神經保護作用。三硝基丙酸則是一種粒線體抑制劑,並且常被用來作為模擬但丁頓舞蹈症(Huntington’s disease)的藥物模型。
目標與假設:探討SHH對於對抗粒線體抑制劑三硝基丙酸毒性之神經保護作用並探討其機制。
材料與方法:在初代培養的18天胎鼠大腦皮質神經細胞中,以免疫螢光染色、西方墨點法等技術分別觀察細胞的存活率和型態及相對蛋白質的表達量。
結果:神經細胞外給SHH具生物活性片段(以下簡稱SHH-N)之前處理(3 ng/ml、6小時)可對三硝基丙酸(2.5 mM、16小時)之神經毒性造成保護作用。此外,外給SHH-N可以刺激神經細胞中extracellular signal-regulated kinase-1/2 (ERK-1/2)與Janus kinase-2 (JAK-2)的磷酸化以及特定蛋白質如neuronal nitric oxide synthase (nNOS)、cGMP-dependent protein kinase (PKG)以及nuclear factor-kappaB (NF-kB)次單元蛋白質p65與p50的表現量增加。而SHH-N之神經保護作用也可以被ERK1/2抑制劑PD98059、NOS抑制劑L-NAME、PKG-1抑制劑KT5823、guanylyl cyclase抑制劑ODQ、JAK-2抑制劑AG490、以及NF-kB抑制劑SN50所阻斷。
結論:外給SHH-N可以保護神經細胞對抗三硝基丙酸的毒性,而這樣的神經保護機制可能與其刺激ERK1/2、nNOS、PKG-1/-2表達或增加JAK-2與NF-kB活性有部分的相關性。
Backgrounds and hypothesis: Sonic hedgehog (SHH) is a crucial regulator during vertebrate embryogenesis. Recent studies showed that SHH, in addition to being a morphogen, may also provide neuroprotective effect towards neuron. 3-Nitropropionic acid (3-NP) is an irreversible mitochondrial inhibitor commonly used as a pharmacological model to mimic the pathogenic mechanisms of Huntington’s disease (HD). However, whether SHH may confer neuronal resistance to cultured cortical neurons against mitochondrial dysfunction induced by 3-NP exposure has never been studied. In the present study, we hypothesized that preconditioning of cortical neurons may exert neuroprotective actions against mitochondrial inhibition induced by 3-NP.
Materials and methods: Primary cultures of fetal rat cortical neurons were pre-treated with biologically active N-terminal fragment of SHH (SHH-N) followed by challenge with 3-NP. Cell survival was assessed by Hoechst staining, propidium iodide (PI)/Hoechst double staining and immunocytochemistry. Protein expression was determined by Western blotting. Pharmacological inhibitors were used to block the biological actions of specific mediators in selective signal transduction pathways.
Results: We found that application of SHH-N was capable of attenuating neurotoxicity caused by 3-NP in a dose- and time-dependent manner. The SHH-N effects were abolished by cyclopamine, an inhibitor of the canonical SHH signaling pathway. SHH-N appeared to induce the phosphorylation of extracellular signal-regulated kinase-1/2 (ERK-1/2) and Janus kinase-2 (JAK-2). SHH-N also enhanced the expression of neuronal nitric oxide synthase (nNOS), cGMP-dependent protein kinase (PKG), as well as nuclear factor-kappaB (NF-kB) subunits p65 and p50. Consistently, the SHH-N-dependent attenuation of 3-NP toxicity was neutralized by PD98059, AG490, L-NAME, KT5823, and SN50, the respective inhibitor of ERK1/2, JAK-2, NOS, PKG, and NF-kB. The SHH-N effect was also abolished by guanylyl cyclase inhibitor ODQ.
Conclusions: Based on these results, we conclude that SHH may protect cultured rat cortical neurons against mitochondrial dysfunction induced by 3-NP; furthermore, the neuroprotective effects of SHH may be in part mediated by induction of ERK1/2, NOS, PKG, JAK-2, and activation of NF-kB.
論文電子檔著作權授權書……………………………………………………… i
論文審定同意書………………………………………………………………… ii
誌謝………………………………………………………………………………. iii
中文摘要………………………………………………………………………… iv
英文摘要…………………………………………………………………………. vi
目錄………………………………………………………………………………. viii
圖表目錄…………………………………………………………………………. x
第一章背景…………………………………………………………………..... 1
1.1但丁頓舞蹈症與三硝基丙酸…………………………………..………. 2
1.2 Sonic Hedgehog………………………………………………………… 4
第二章材料與方法……………………………………………………………. 6
2.1 藥劑…………………………………………………………………….. 7
2.2 初代皮質細胞………………………………………………………….. 7
2.3 細胞存活分析………………………………………………………….. 8
2.4 細胞螢光免疫染色…………………………………………………….. 9
2.5 西方墨點法…………………………………………………………….. 10
2.6 統計…………………………………………………………………….. 12
結果………………………………………………………………………………. 13
討論………………………………………………………………………………. 21
參考文獻…………………………………………………………………………. 26
附錄………………………………………………………………………………. 35
Signature Page ………………………………….………………………………… i
Thesis Approval Form ……………………………………………………………. ii
Acknowledgments ………………………………………………………………… iii
Chinese Abstract ……………………………...…………………………………... iv
English Abstract ………………..…………………………………………………. vi
Table of Contents …………………………………………………………………. viii
List of Figures …………………………………………………………………….. x
Chapter 1 Intorduction…………………………………………………………...... 1
1.1 Huntington’s disease and 3-Nitropropionic acid………………………. 2
1.2 Sonic hedgehog………………………………………………………… 4
Chapter 2 Materials and methods…………………………………………………. 6
2.1 Reagents………………………………………………………………... 7
2.2 Primary neuronal cultures……………………………………………… 7
2.3 Cell survival assays by Hoechst staining and PI/Hoechst double staining…………………………………………………………………. 2.4 Immunocytochemistry by confocal microscopy and Fluorescence Microscope……………………………………………………………... 2.5 Western blotting………………………………………………………... 8 9 10
2.6 Statistical analysis……………………………………………………… 12
Results…………………………………………………………………................... 13
Discussion ………………………………………………………………………… 21
References ………………………………………………………………………… 26
Appendices ………………………………………………………………………... 35
Agouni, A., Mostefai, H. A., Porro, C., Carusio, N., Favre, J., Richard, V., Henrion, D., Martinez, M. C. and Andriantsitohaina, R. (2007) Sonic hedgehog carried by microparticles corrects endothelial injury through nitric oxide release. FASEB J, 21, 2735-2741.
Ahn, S. and Joyner, A. L. (2005) In vivo analysis of quiescent adult neural stem cells responding to Sonic hedgehog. Nature, 437, 894-897.
Alston, T. A., Mela, L. and Bright, H. J. (1977) 3-Nitropropionate, the toxic substance of Indigofera, is a suicide inactivator of succinate dehydrogenase. Proc Natl Acad Sci U S A, 74, 3767-3771.
Barnes, G. T., Duyao, M. P., Ambrose, C. M., McNeil, S., Persichetti, F., Srinidhi, J., Gusella, J. F. and MacDonald, M. E. (1994) Mouse Huntington's disease gene homolog (Hdh). Somat Cell Mol Genet, 20, 87-97.
Beal, M. F. (1998) Mitochondrial dysfunction in neurodegenerative diseases. Biochim Biophys Acta, 1366, 211-223.
Besshoh, S., Bawa, D., Teves, L., Wallace, M. C. and Gurd, J. W. (2005) Increased phosphorylation and redistribution of NMDA receptors between synaptic lipid rafts and post-synaptic densities following transient global ischemia in the rat brain. J Neurochem, 93, 186-194.
Bonthius, D. J., Bonthius, N. E., Li, S. and Karacay, B. (2008) The protective effect of neuronal nitric oxide synthase (nNOS) against alcohol toxicity depends upon the NO-cGMP-PKG pathway and NF-kappaB. Neurotoxicology, 29, 1080-1091.
Bonthius, D. J., Luong, T., Bonthius, N. E., Hostager, B. S. and Karacay, B. (2009) Nitric oxide utilizes NF-kappaB to signal its neuroprotective effect against alcohol toxicity. Neuropharmacology, 56, 716-731.
Calabresi, P., Gubellini, P., Centonze, D., Sancesario, G., Morello, M., Giorgi, M., Pisani, A. and Bernardi, G. (1999) A critical role of the nitric oxide/cGMP pathway in corticostriatal long-term depression. J Neurosci, 19, 2489-2499.
Canals, S., Casarejos, M. J., de Bernardo, S., Rodriguez-Martin, E. and Mena, M. A. (2001) Glutathione depletion switches nitric oxide neurotrophic effects to cell death in midbrain cultures: implications for Parkinson's disease. J Neurochem, 79, 1183-1195.
Chen, J. K., Taipale, J., Cooper, M. K. and Beachy, P. A. (2002) Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened. Genes Dev, 16, 2743-2748.
Chiang, C., Litingtung, Y., Lee, E., Young, K. E., Corden, J. L., Westphal, H. and Beachy, P. A. (1996) Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature, 383, 407-413.
Chiche, J. D., Schlutsmeyer, S. M., Bloch, D. B., de la Monte, S. M., Roberts, J. D., Jr., Filippov, G., Janssens, S. P., Rosenzweig, A. and Bloch, K. D. (1998) Adenovirus-mediated gene transfer of cGMP-dependent protein kinase increases the sensitivity of cultured vascular smooth muscle cells to the antiproliferative and pro-apoptotic effects of nitric oxide/cGMP. J Biol Chem, 273, 34263-34271.
Christensen, C., Zhang, S. and Roelink, H. (2006) Inhibition of cGMP-dependent protein kinase reduces the response to sonic hedgehog in neuralized embryoid bodies. Stem Cells Dev, 15, 647-654.
Culotta, E. and Koshland, D. E., Jr. (1992) NO news is good news. Science, 258, 1862-1865.
Dass, B., Iravani, M. M., Huang, C., Barsoum, J., Engber, T. M., Galdes, A. and Jenner, P. (2005) Sonic hedgehog delivered by an adeno-associated virus protects dopaminergic neurones against 6-OHDA toxicity in the rat. J Neural Transm, 112, 763-778.
Dawson, T. M., Sasaki, M., Gonzalez-Zulueta, M. and Dawson, V. L. (1998) Regulation of neuronal nitric oxide synthase and identification of novel nitric oxide signaling pathways. Prog Brain Res, 118, 3-11.
Deckel, A. W. (2001) Nitric oxide and nitric oxide synthase in Huntington's disease. J Neurosci Res, 64, 99-107.
Digicaylioglu, M. and Lipton, S. A. (2001) Erythropoietin-mediated neuroprotection involves cross-talk between Jak2 and NF-kappaB signalling cascades. Nature, 412, 641-647.
Gudi, T., Casteel, D. E., Vinson, C., Boss, G. R. and Pilz, R. B. (2000) NO activation of fos promoter elements requires nuclear translocation of G-kinase I and CREB phosphorylation but is independent of MAP kinase activation. Oncogene, 19, 6324-6333.
Gudi, T., Lohmann, S. M. and Pilz, R. B. (1997) Regulation of gene expression by cyclic GMP-dependent protein kinase requires nuclear translocation of the kinase: identification of a nuclear localization signal. Mol Cell Biol, 17, 5244-5254.
Gutierrez-Frias, C., Sacedon, R., Hernandez-Lopez, C., Cejalvo, T., Crompton, T., Zapata, A. G., Varas, A. and Vicente, A. (2004) Sonic hedgehog regulates early human thymocyte differentiation by counteracting the IL-7-induced development of CD34+ precursor cells. J Immunol, 173, 5046-5053.
He, B. and Weber, G. F. (2003) Phosphorylation of NF-kappaB proteins by cyclic GMP-dependent kinase. A noncanonical pathway to NF-kappaB activation. Eur J Biochem, 270, 2174-2185.
Hofmann, F., Ammendola, A. and Schlossmann, J. (2000) Rising behind NO: cGMP-dependent protein kinases. J Cell Sci, 113, 1671-1676.
Ju, T. C., Chen, S. D., Liu, C. C. and Yang, D. I. (2005) Protective effects of S-nitrosoglutathione against amyloid beta-peptide neurotoxicity. Free Radic Biol Med, 38, 938-949.
Ju, T. C., Yang, Y. T. and Yang, D. I. (2004) Protective effects of S-nitrosoglutathione against neurotoxicity of 3-nitropropionic acid in rat. Neurosci Lett, 362, 226-231.
Kusano, K. F., Pola, R., Murayama, T. et al. (2005) Sonic hedgehog myocardial gene therapy: tissue repair through transient reconstitution of embryonic signaling. Nat Med, 11, 1197-1204.
Loweth, A. C., Williams, G. T., Scarpello, J. H. and Morgan, N. G. (1997) Evidence for the involvement of cGMP and protein kinase G in nitric oxide-induced apoptosis in the pancreatic B-cell line, HIT-T15. FEBS Lett, 400, 285-288.
Miao, N., Wang, M., Ott, J. A., D'Alessandro, J. S., Woolf, T. M., Bumcrot, D. A., Mahanthappa, N. K. and Pang, K. (1997) Sonic hedgehog promotes the survival of specific CNS neuron populations and protects these cells from toxic insult In vitro. J Neurosci, 17, 5891-5899.
Palfi, S., Ferrante, R. J., Brouillet, E., Beal, M. F., Dolan, R., Guyot, M. C., Peschanski, M. and Hantraye, P. (1996) Chronic 3-nitropropionic acid treatment in baboons replicates the cognitive and motor deficits of Huntington's disease. J Neurosci, 16, 3019-3025.
Panov, A. V., Gutekunst, C. A., Leavitt, B. R., Hayden, M. R., Burke, J. R., Strittmatter, W. J. and Greenamyre, J. T. (2002) Early mitochondrial calcium defects in Huntington's disease are a direct effect of polyglutamines. Nat Neurosci, 5, 731-736.
Perez-Severiano, F., Escalante, B., Vergara, P., Rios, C. and Segovia, J. (2002) Age-dependent changes in nitric oxide synthase activity and protein expression in striata of mice transgenic for the Huntington's disease mutation. Brain Res, 951, 36-42.
Podlasek, C. A., Meroz, C. L., Korolis, H., Tang, Y., McKenna, K. E. and McVary, K. T. (2005) Sonic hedgehog, the penis and erectile dysfunction: a review of sonic hedgehog signaling in the penis. Curr Pharm Des, 11, 4011-4027.
Riobo, N. A., Lu, K., Ai, X., Haines, G. M. and Emerson, C. P., Jr. (2006) Phosphoinositide 3-kinase and Akt are essential for Sonic Hedgehog signaling. Proc Natl Acad Sci U S A, 103, 4505-4510.
Ruiz i Altaba, A., Sanchez, P. and Dahmane, N. (2002) Gli and hedgehog in cancer: tumours, embryos and stem cells. Nat Rev Cancer, 2, 361-372.
Shiojima, I. and Walsh, K. (2002) Role of Akt signaling in vascular homeostasis and angiogenesis. Circ Res, 90, 1243-1250.
The Huntington's Disease Collaborative Research Group. (1993) A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes.. Cell, 72, 971-983.
Tabrizi, S. J., Workman, J., Hart, P. E., Mangiarini, L., Mahal, A., Bates, G., Cooper, J. M. and Schapira, A. H. (2000) Mitochondrial dysfunction and free radical damage in the Huntington R6/2 transgenic mouse. Ann Neurol, 47, 80-86.
Taimor, G., Hofstaetter, B. and Piper, H. M. (2000) Apoptosis induction by nitric oxide in adult cardiomyocytes via cGMP-signaling and its impairment after simulated ischemia. Cardiovasc Res, 45, 588-594.
Takuma, K., Phuagphong, P., Lee, E., Mori, K., Baba, A. and Matsuda, T. (2001) Anti-apoptotic effect of cGMP in cultured astrocytes: inhibition by cGMP-dependent protein kinase of mitochondrial permeable transition pore. J Biol Chem, 276, 48093-48099.
Weber, G. F., Abromson-Leeman, S. and Cantor, H. (1995) A signaling pathway coupled to T cell receptor ligation by MMTV superantigen leading to transient activation and programmed cell death. Immunity, 2, 363-372.
Wu, C. L., Chen, S. D., Hwang, C. S. and Yang, D. I. (2009a) Sonic hedgehog mediates BDNF-induced neuroprotection against mitochondrial inhibitor 3-nitropropionic acid. Biochem Biophys Res Commun, 385, 112-117.
Wu, C. L., Hwang, C. S. and Yang, D. I. (2009b) Protective effects of brain-derived neurotrophic factor against neurotoxicity of 3-nitropropionic acid in rat cortical neurons. Neurotoxicology, 30, 718-726.
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