Protein phosphatase 2A (PP2A)是屬於serine/threonine去磷酸酶的一種，能調控細胞的生長、存活和凋亡。哺乳類動物中，Bβ次單位是PP2A在腦神經細胞中的專一表現的次表型。當Bβ基因有所損害或過度表現時，會造成神經細胞的病變。本論文分別將能專一表現於細胞質的Bβ1和專一表現於粒線體的Bβ2質體轉殖入神經腫瘤母細胞SK-N-SH中，建立穩定細胞株。利用H2O2和tert-butyl hydroperoxide (tBHP)作為氧化壓力來源，經48小時的處理後，Bβ2細胞株的存活率會急遽下降，由Annexin V和PI雙重染色的結果中證實是因細胞凋亡而降低存活率，但Bβ1細胞株則否。進一步轉殖LC3-EGFP質體和對酸性液泡胞器染色鑑定細胞自噬的發生，可以看出Bβ2細胞株本身便有些微細胞自噬體存在，當經氧化壓力後更引發Bβ2細胞株有更多細胞自噬體出現。但以細胞自噬抑制劑3-methyladenine (3-MA)對細胞作前處理24小時後，再經過氧化物處理，則各Bβ2細胞株的sub-G1期細胞比例皆會下降，而酸性液泡胞器的密度也明顯降低。證明氧化壓力所引起的細胞凋亡是經由細胞自噬路徑而產生。另利用降低培養液中的血清濃度模擬營養缺乏的情況，當無血清培養48小時的Bβ1細胞株之sub-G1期細胞比例較Bβ2細胞株高出許多，當血清濃度降低時，Bβ1細胞株的酸性液泡胞器也較Bβ2細胞株的密度高，證實營養缺乏在Bβ1細胞株所引發的細胞凋亡是經由細胞自噬。本論文針對過度表現PPP2R2B的細胞株分別於氧化壓力和營養缺乏的壓力下，細胞所產生的生理變化，模擬神經退化病變的產生，並瞭解病變機制，祈能對神經退化疾病的研究與治療有所助益。

Protein phosphatase 2A (PP2A) is one of four major classes of serine/ threoine phosphatases. PP2A regulates in many cell functions, including growth, survival, and apoptosis. In mammals, the B family Bβ encodes a neuron-specific member of PP2A subunit with distinct expression patterns in brain. The defective and overexpressed Bβ can be associated with initiation of neurodegeneration. In this work, the construct of B subunit encoding cytoplasm-specific Bβ1 and mitochondria-specific Bβ2 was transfected into neuroblastoma cells, SK-N-SH, respectively, and the stable clones over-expressing either Bβ1 or Bβ2 were established. The cells were stimulated with oxidative pressure, H2O2 and tert-butyl hydroperoxide (t-BHP). Both H2O2 and tBHP reduced cell viabilities the Bβ2 clones after 48h, but not in Bβ1 clone. By Annexin V and propidium iodide staining, we have found that the apoptotic cell death in Bβ2 clones were increased in H2O2 and in tBHP depending on the clones used; whereas Bβ1 clones and the parental cells were unaffected. Using transient cells expressing autophagic marker, green fluorescent protein-tagged microtubule-associated protein light chain 3 (EGFP–LC3) and staining acidic vesicular organelles, we found that the induction of autophagy in Bβ2 clones is associated with apoptotic cell death under oxidative stress. The autophagy inhibitor, 3-methyladenine (3-MA), inhibits apoptosis and decreased acidic vesicular organelles induced by oxidative stress. On the other hand, compared to SK-N-SH cells, Bβ1 clones are more sensitive to serum deprivation by increasing sub-G1 population, while Bβ2 clones were less sensitive. This study offers more in-death knowledge on physiological changes of neuron cells overexpressing PPP2R2B under oxidative stress and nutrition deprivation.

伍、參考文獻：

ASH Image Bank, http://ashimagebank.hematologylibrary.org/cgi/content-nw/full/ 2001/1205/100226/3, (2001).

Abraham, D., Podar, K., Pacher, M., Kubicek, M., Welzel, N., Hemmings, B. A., Dilworth, S. M., Mischak, H., Kolch, W. & Baccarini, M. Raf-1-associated Protein Phosphatase 2A as a Positive Regulator of Kinase Activation. J. Biol. Chem. (2000) 275, 22300–22304.

Alessi, D. R., Gomez, N., Moorhead, G., Lewis, T., Keyse, S. M. & Cohen, P. Inactivation of p42 MAP kinase by protein phosphatase 2A and a protein tyrosine phosphatase, but not CL100, in various cell lines. Curr. Biol. (1995) 5, 283–295.

Amoroso, S., Gioielli, A., Cataldi, M., Di Renzo, G., Annunziato, L. In the neuronal cell line SH-SY5Y, oxidative stress-induced free radical overproduction causes cell death without any participation of intracellular Ca(2+) increase. Biochim. Biophys. Acta. (1999) 1452, 151–160.

Andjelkovic, M., Jakubowicz, T., Cron,P., Ming, X. F., Han, J. W. and Hemmings, B. A. Activation and phosphorylation of a pleckstrin homology domain containing protein kinase (RAC-PK/PKB) promoted by serum and protein phosphatase inhibitors. PNAS. (1996) 93, 5699-5704.

Arendt, T., Holzer, M., Fruth, R., Bruckner, M. K., and Gartner, U. Phosphorylation of tau, Abeta-formation, and apoptosis after in vivo inhibition of PP-1 and PP-2A. Neurobiol. Aging (1998) 19, 3-13.

Arico, S., Petiot, A., Bauvy, C., Dubbelhuis, P. F., Meijer, A. J., Codogno, P. and Ogier-Denis. The Tumor suppressor PTEN postively regulates macroautophagy by inhibiting the phosphatidylinonsitol 3-kinase/protein kinase B pathway. J. Biol. Chem. (2001) 276, 35243-35246.

Bratton, S. B., MacFarlane, M., Cain, K., and Cohen, G. M. Protein complexes activate distinct caspase cascades in death receptor and stressinduced apoptosis. Exp. Cell Res. (2000) 256, 27- 33.

Bratton, S. B., and Cohen, G. M. Apoptotic death sensor: an organelle’s alter ego? Trends Pharmacol. Sci. (2001) 22,306-315.

Charles, I., Khalyfa, A., Kumar, D. M., Krishnamoorthy, R. R., Roque, R. S., Cooper, N., and Agarwal, N. Serum deprivation induces apoptotic cell death of transformed rat retinal ganglion cells via mitochondrial signaling pathways. Invest. Ophthalmol. Vis. Sci. (2005) 46, 1330-1338.

Charlesene, M. Blue., Barbara, A., Wetmorea, J. F., Sanchezb, W. J., Freed, B., and Alex, M. Apoptosis mediated by p53 in rat neural AF5 cells following treatment with hydrogen peroxide and staurosporine. Brain Res. (2006) 1112, 1-15.

Chiang, C. W., Harris, G., Ellig, C., Masters, S. C., Subramanian, R., Shenolikar, S., Wadzinski, B. E., and Yang, E. Protein phosphatase 2A activates the proapoptotic function of BAD in interleukin-3-dependent lymphoid cells by a mechanism requiring 14-3-3 dissociation. Blood (2001) 97, 1289-1297.

Choi, J., Sullards, M. C., Olzmann, J. A., Rees, H. D.,Weintraub, S. T., Bostwick, D. E., Gering, M., Levey, A. I., and Chin, L. S. Oxidative damage of DJ-1 is linked to sporadic Parkinson and Alzheimer disease. J. Biol. Chem. (2006) 281, 10816-10824.

Dagda, R. K., Zaucha, J. A., Wadzinski, B. E., and Strack, S. A developmentally regulated, neuron-specific splice variant of the variable subunit B beta targets protein phosphatase 2A to mitochondria and modulates apoptosis. J. Biol. Chem. (2003) 278, 24976-24985.

Deng, X., Ito, T., Carr, B., Mumby, M., and May Jr., W. S. Reversible phosphorylation of Bcl-2 following interleukin 3 or bryostatin 1 is mediated by direct interaction with protein phosphatase 2A. J. Biol. Chem. (1998) 273, 34157-34163.

Droge, W. Oxidative aging and insulin receptor signaling. J. Gerontol. A, Biol. Sci. Med. Sci. (2005) 60, 1378–1385.

Dunn, W. A. Jr. Autophagy and related mechanisms of lysosome-mediated protein degradation. Trends Cell Biol. (1994) 4, 139-143.

Finkel, T., and Holbrook, N.J. Oxidants, oxidative stress and the biology of ageing. Nature (2000) 408, 239–247.

Fujita, R., Yoshida, A., Mizuno, K, and Ueda, H. Cell density-dependent death mode switch of cultured cortical neurons under serum-free starvation stress. Cell Mol. Neurobiol. (2001) 4, 317-324.

Galadari, S., Kishikawa, K., Kamibayashi, C., Mumby, M. C., and Hannun, Y. A. Purification and characterization of ceramide-activated protein phosphatases. Biochem. (1998) 37, 11232-11238.

Gerlier, D. and Thomasset, N. Use of MTT colorimetric assay to measure cell activation. J. Immunol. Methods (1986) 94, 57-63.

Gorman, A.M., McGowan, A., O'Neill, C., and Cotter, T. Oxidative stress and apoptosis in neurodegeneration. J. Neurol. Sci. (1996) 139, 45–52.

Gong, C. X., Lidsky, T., Wegiel, J., Zuck, L., Grundke-Iqbal, I., and Iqbal, K. Phosphorylation of microtubule-associated protein tau is regulated by protein phosphatase 2A in mammalian brain. Implications for neurofibrillary degeneration in Alzheimer's disease. J. Biol. Chem. (2000) 275, 5535-5544.

Götz, J. Tau and transgenic animal models. Brain Res. Brain Res. Rev. (2001) 35, 266–286.

Haccard, O., Jessus, C., Cayla, X., Goris, J., Merlevede, W. & Ozon, R. In vivo activation of a microtubule-associated protein kinase during meiotic maturation of the Xenopus oocyte. Eur. J. Biochem. (1990) 192, 633–642.

Hacker, G. The morphology of apoptosis. Cell Tissue Res. (2000) 301, 5 -17.

Hamasaki, M., Noda, T., Baba, M., and Ohsumi, Y. Starvation triggers the delivery of the endoplas¬mic reticulum to the vacuole via autophagy in yeast. Traffic (2005) 6, 56 65.

Hara, T., Nakamura, K., Matsui, M., Yamamoto, A., Nakahara, Y., Suzuki-Migishima, R., Yokoyama, M., Mishima, K., Saito, I., Okano, H., and Mizushima, N. Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice. Nature (2006) 441, 885-889.

Harding, T. M., A. Hefner-Gravink, M. Thumm, and D. J. Klionsky. Genetic and phenotypic overlap between autophagy and the cytoplasm to vacuole protein targeting pathway. J. Biol. Chem. (1996) 271, 17621–17624.

Halliwell, B., and Guttericge, J. M. C. Oxygen toxicity, oxygen radicals, transition metal and diseases. Biochem. J. (1984) 219, 1-5.

Halliwell, B., and Aruoma, O. I. DNA damage by oxygen-derived species. Its mechanism and measurement in mammalian systems. FEBS Lett. (1991) 281, 9–19.

Hershko, A. and Ciechanover, A. The ubiguitin sysyem. Annu. Rev. Bioc. (1998) 67, 425-479.

Holmes, S. E., O'Hearn, E. E., McInnis, M. G., Gorelick-Feldman, D. A., Kleiderlein, J. J., Callahan, C., Kwak, N. G., Ingersoll-Ashworth, R. G., Sherr, M., Sumner, A. J., Sharp, A. H., Ananth, U., Seltzer, W. K., Boss, M. A., Vieria-Saecker, A. M., Epplen, J. T., Riess, O., Ross, C. A., and Margolis, R. L. Expansion of a novel CAG trinucleotide repeat in the 5' region of PPP2R2B is associated with SCA12. Nature Geneicst. (1999) 23, 391-392.

Hutchins, M. U., M. Veenhuis, and D. J. Klionsky. Peroxisome degradation in Saccharomyces cerevisiae is dependent on machinery of macroautophagy and the Cvt pathway. J. Cell Sci. (1999) 112, 4079–4087.

Jang, M., Cai, L., Udeani, G. O., Slowing, K. V., Thomas, C. F., Beecher, C. W.,Fong, H. H., Farnsworth, N. R., Kinghorn, A. D., Mehta, R. G., Moon, R. C., and Pezzuto, J. M. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science (1997) 275, 218–220.

Kabeya, Y., Mizushima, N., Ueno, T., Yamamoto, A., Kirisako, T., Noda, T., Kominami, E., Ohsumi, Y., and Yoshimori, T. LC3, a mammalian homologue of yeast Apg8p is localized in autophagosome membranes affter processing. EMBO J. (2000) 19, 5720-5728.
Kleinberger, T., and Shenk, T. Adenovirus E4orf4 protein binds to protein phosphatase 2A, and the complex down regulates E1A-enhanced junB transcription. J. Virol. (1993) 67, 7556-7560.

Klumpp, S. and Krieglstein, J. Serine/threonine protein phosphatases in apoptosis. Curr. Opin. Pharmacol. (2002) 2, 458-462.

Komatsu, M., Waguri, S., Chiba, T., Murata, S., Iwata, J. I., Tanida, I., Ueno, T., Koike, M., Uchiyama, Y., Kominami, E., and Tanaka, K. Loss of autophagy in the central nervous system causes neurodegeneration in mice. Nature (2006) 441, 880-884.

Krishnamurthy, P. K., Mays, J. L., Bijur, G. N., and Johnson, G. V. W. Transient oxidative stress in SH-SY5Y human neuroblastoma cells results in caspase dependent and independent cell death and tau proteolysis. J. Neurosci. Res. (2000) 61, 515–523.

Kunchithapautham, K. and Rohrer, B. Apoptosis and autophagy in photoreceptors exposed to oxidative stress. Autophagy (2007) 3, 433-441.

Levine, B., and Klionsky, D. J. Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev. Cell (2004) 6, 463-477.

Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, E. S., and Wang, X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase 9 complex initiates an apoptotic protease cascade. Cell (1997) 91, 479-489.

Liang, X. H., Jackson, S., Seaman, M., Brown, K., Kempkes, B., Hibshoosh, H. and Levine, B. Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature (1999) 402, 672-676.

Liao, Y. and Hung, M. C. A new role of protein phosphatase 2A in adenoviral E1A protein-mediated sensitization to anticancer drug-induced apoptosis in human breast cancer cells. Cancer Res. (2004) 64, 5938-5942.

Lin, H. J., Wang, X., Shaffer, K. M., Sasaki, C. Y., and Ma, W. Characterization of H2O2-induced acute apoptosis in cultured neural stem/progenitor cells. FEBS Lett. (2004) 570, 102-106.
Liu, H., Miller, E., van de Water, B., and Stevens, J. L. Endoplasmic reticulum stress proteins block oxidant-induced Ca21 increases and cell death. J. Biol. Chem. (1998) 273, 12858 –12862.

Markesbery, W. R. Oxidative stress hypothesis in Alzheimer's disease. Free Radic. Biol. Med. (1997) 23, 134–147.

Masaki, N., Kyle, M. E., Serroni, A., and Farber, J. L. Mitochondrial damage as a mechanism of cell injury in the killing of cultured hepatocytes by tert-butyl hydroperoxide. Arch. Biochem. Biophys. (1989) 270, 672–680.

Mayer, R. E., Hendrix, P., Cron, P., Matthies, R., Stone, S. R., Goris, J., Merlevede, W., Hofsteenge, J., and Hemmings, B. A. Structure of the 55-kDa regulatory subunit of protein phosphatase 2A: evidence for a neuronal-specific isoform. Biochem. (1991) 30, 3589-3597.

Millward, T. A., Zolnierowicz, S., and Hemmings, B. A. Regulation of protein kinase cascades by protein phosphatase 2A. Trends Biochem. Sci. (1999) 24, 186-191.

Moran, M. F., Koch, C. A., Anderson, D., Ellis, C., England, L., Martin, G. S. & Pawson, T. Src homology region 2 domains direct protein-protein interactions in signal transduction. Proc. Natl. Acad. Sci. USA (1990) 87, 8622–8626.

Nicotera, P., McConkey, D., Svensson, S. A., Bellomo, G., Orrenius, S. Correlation between cytosolic Ca21 concentration and cytotoxicity in hepatocytes exposed to oxidative stress. Toxicology (1988) 52, 55– 63.

Rathmell, J. C., and Thompson, C. B. Pathways of apoptosis in lymphocyte development, homeostasis, and disease. Cell (2002) 109, 97-107.

Reggiori, F. and Klionsky D. J. Autophagy in the eukaryotic cell. Eukaryotic Cell. (2002) 1, 11-21.

Resjö, S., Göransson, O., Härndahl, L., Zolnierowicz, S., Manganiello, V. and Degerman, E. Protein phosphatase 2A is the main phosphatase involved in the regulation of protein kinase B in rat adipocytes. Cellular Signalling (2002) 14, 231-238.

Rhee, S.G. Redox signaling: hydrogen peroxide as intracellular messenger. Exp. Mol. Med. (1999) 31, 53-59.

Rich, K. A., Burkett, C., and Webster, P. Cytoplasmic bacteria can be targets for autophagy. Cell. Microbiol. (2003) 5, 455-468.

Santoro, M. F., Annand, R. R., Robertson, M. M., Peng, Y. W., Brady, M. J., Mankovich, J. A., Hackett, M. C., Ghayur, T., Walter, G., Wong, W. W., and Giegel, D. A. Regulation of protein phosphatase 2A activity by caspase- 3 during apoptosis. J. Biol. Chem. (1998) 273, 13119-13128.

Sasahara, Y., Kobayashi, T., Onodera, H., Onoda, M., Ohnishi, M., Kato, S., Kusuda, K., Shima, H., Nagao, M., Abei, H., Yanagawa, Y. Hiraga, A., and Tamura, S. Okadaic acid suppresses neural differentiation-dependent expression of the neurofilament-L gene in P19 embryonal carcinoma cells by post-transcriptional modification. J. Biol. Chem. (1996) 271, 25950-25957.

Schippling, S., Kontush, A., Arlt, S., Buhmann, C., Sturenburg, H., Mann, U., Muller-Thomsen, T., and Beisiegel , U. Increased lipoprotein oxidation in Alzheimer's disease. Free Radic. Biol. Med. (2000) 28, 351–360.

Scott, S. V., A. Hefner-Gravink, K. A. Morano, T. Noda, Y. Ohsumi, and D. J. Klionsky. Cytoplasm-to-vacuole targeting and autophagy employ the same machinery to deliver proteins to the yeast vacuole. Proc. Natl. Acad. Sci. USA. (1996) 93, 12304–12308.

Seglen, P. O. and Gordon, P. B. 3-Methyladenine: Specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc. Natl Acad. Sci. USA (1982) 79, 1889–1892.

Sontag, E., Nunbhakdi-Craig, V., Lee, G., Bloom, G. S., and Mumby, M. C. Regulation of the phosphorylation state and microtubule-binding activity of Tau by protein phosphatase 2A. Neuron (1996) 17, 1201-1207.

Stennicke, H. R., and Salvessen, G. S. Properties of caspases. Biochim. Biophys. Acta (1998) 1387, 17- 31.

Strack, S., Zaucha, J. A., Ebner, F. F., Colbran, R. J., and Wadzinski, B. E. Brain protein phosphatase 2A: developmental regulation and distinct cellular and subcellular localization by B subunits. J. Comp. Neurol. (1998) 392, 515-527.

Strack, S., Ruediger, R., Walter, G., Dagda, R. K., Barwacz, C. A., and Cribbs, J. T. Protein phosphatase 2A holoenzyme assembly: identification of contacts between B-family regulatory and scaffolding A subunits. J. Biol. Chem. (2002) 277, 20750-20755.

Sun, L., Liu, S. Y., Zhou, X. W., Wang, X. C., Liu, R., Wang, Q., and Wang, J. Z. Inhibition of protein phosphatase 2A- and protein phosphatase 1-induced tau hyperphosphorylation and impairment of spatial memory retention in rats. Neuroscience (2003) 118, 1175-1182.

Talanian, R. V., Quinlan, C., Trautz, S., Hackett, M. C., Mankovich, J. A., Banach, D., Ghayur, T., Brady, K. D., and Wong, W. W. Substrate specificities of caspase family proteases. J. Biol. Chem. (1997) 272, 9677-9682.

Virshup, D. M. Protein phosphatase 2A: a panoply of enzymes. Curr Opin Cell Biol. (2000) 12, 180-185.

Walczak, H., and Krammer, P. H. The CD 95 (APO-1/Fas) and the TRAIL (APO/2L) apoptosis system. Exp. Cell Ras. (2000) 256, 58-66.

Wang, K. K., and Yuen, P. W. Calpain inhibition: an overview of its therapeutic potential. Trends Pharmacol. Sci. (1994) 15, 412– 419.

Yoshikawa, A., Saito, Y., and Maruyama, K. Lignan compounds and 4,4’-dihydroxybiphenyl protect C2C12 cells against damage from oxidative stress. Biochem. Biophys. Res. Commun (2006) 344, 394-399.

Zhang, L., Zhao, B., Yew, D. T., Kusiak, J. W., and Roth, G. S. Processing of Alzheimer’s amyloid precursor protein during H2O2-induced apoptosis in human neuronal cells. Biochem. Biophys. Res. Commun (1997) 235, 845-848.

Zhang, L., Yu, H., Sun, Y., Lin, X., Chen, B., Tan, C., Cao, G., and Wang, Z. Protective effects of salidroside on hydrogen peroxide-induced apoptosis in SH-SY5Y human neuroblastoma cells. Eur. J. Pharmacol. (2007) 564, 18-25.
Zhao, K., Luo, G., Giannelli, S., and Szeto H. H. Mitochondria-targeted peptide prevents mitochondrial depolarization and apoptosis induced by tert-butyl hydroperoxide in neuronal cell lines. Biochem. Pharmacol. (2005) 70, 1796–1806.