Protein phosphatase 2A (簡稱PP2A)是細胞內重要的蛋白去磷酸化酵素，其次單元B調控PP2A在細胞內作用的位置及催化的受質種類。PPP2R2B是表現在腦神經細胞中的PP2A調控次單元B。腦部表現的PP2A調節tau蛋白的磷酸化。Tau蛋白過度磷酸化和PP2A活性下降，皆和阿茲海默氏症(AD)相關。本實驗室的研究亦發現PPP2R2B的低啟動子活性，和國人的阿茲海默氏症顯著相關。本研究第一部份是以PPP2R2B啟動子接上EGFP報導基因，來建立人類胚胎腎HEK-293及神經腫瘤SK-N-SH細胞，轉染入轉錄因子SP1及CREB1後，分析綠螢光量變化，並未呈現預期的調控情形。第二部分為探討PPP2R2B DNA甲基化的外遺傳調控，與阿茲海默氏症的相關性。選取五組年齡及性別配對的病人及正常人DNA樣品，經Bisulfite定序，結果發現AD病人的PPP2R2B的5'端甲基化程度有高於正常人的趨勢，尤其是-311及-310的位置，雖然並沒有到達顯著差異。進一步的神經(SK-N-SH、SH-SY5Y)及非神經(HEK-293)腫瘤細胞PPP2R2B啟動子定序，顯示HEK-293細胞PPP2R2B啟動子上的甲基化，但以去甲基化藥物5-aza-dC處理HEK-293後，PPP2R2B表現量並未顯著上升，MeCP2的抗體的染色質免疫沈澱亦未看到MeCP2蛋白結合到PPP2R2B啟動子上。

Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase expressed in all eukaryotic cells. The regulatory B subunit confers substrate specificity and sub-cellular localization of the PP2A holoenzyme. PPP2R2B is a regulatory B subunit expressed throughout the brain. The brain specific PPP2R2B regulates PP2A dephosphorylation activity for microtubule-associated protein tau. The association of pathological hyperphosphorylated tau and reduced PP2A activity with Alzheimer's disease (AD) has been established. Our case-control study and reporter assay have also revealed that the rare low transcriptional activity alleles of PPP2R2B are associated with AD. In the first part of the study, PPP2R2B-driven EGFP construct was used to generate human embryonic kidney (HEK)-293 and neuroblastoma SK-N-SH cell lines. SP1 and CREB1 co-transfection did not enhance PPP2R2B expression. In the second part of the study, epigenetic control of DNA methylation affecting AD susceptivity was explored. Bisulfite sequencing was performed to assess the DNA methylation using lymphocyte DNA from five AD patients and age- and gender-matched controls. The results of increased DNA methylation (although not significantly) in the PPP2R2B gene 5' region, especially -311 and -310, suggest that the epigenetic change may alter the PPP2R2B expression in AD patients. Further direct bisulfite-sequencing of HEK-293 cells revealed increased DNA methylation in the PPP2R2B gene 5' region. However, real-time PCR quantitation of 5-aza-dC treated HEK-293 cells did not show increased PPP2R2B expression. Chromatin IP with MeCP2 antibody and PCR also did not support MeCP2 binding to PPP2R2B promoter.

Alzheimer A. Uber eine eigenartige Erkrankung der Hirnrinde. Allg Z Psychiatr 1907;64:146–148.
Berchtold NC, Cotman CW. Evolution in the conceptualization of dementia and Alzheimer's disease: Greco-Roman period to the 1960s. Neurobiol Aging 1998;19:173–189.
Blennow K, de Leon MJ, Zetterberg H. Alzheimer's disease. Lancet 2006;368:387–403.
Chen CM, Hou YT, Liu JY, Wu YR, Lin CH, Fung HC, Hsu WC, Hsu Y, Lee SH, Hsieh-Li HM, Su MT, Chen ST, Lane HY, Lee-Chen GJ. PPP2R2B CAG repeat length in the Han Chinese in Taiwan: Association analyses in neurological and psychiatric disorders and potential functional implications. Am J Med Genet B Neuropsychiatr Genet 2009a;150B:124–129.
Chen KL, Wang SS, Yang YY, Yuan RY, Chen RM, Hu CJ. The epigenetic effects of amyloid-beta(1–40) on global DNA and neprilysin genes in murine cerebral endothelial cells. Biochem Biophys Res Commun 2009b;378:57–61.
Chi P, Allis CD, Wang GG. Covalent histone modifications–miswritten, misinterpreted and mis-erased in human cancers. Nat Rev Cancer 2010;10:457–469.
Corder EH, Saunders AM, Strittmatter WJ, Schmechel DE, Gaskell PC, Small GW, Roses AD, Haines JL, Pericak-Vance MA. Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late onset families. Science 1993;261:921–923.
Dagda RK, Zaucha JA, Wadzinski BE, Strack S. A developmentally regulated, neuron-specific splice variant of the variable subunit Bbeta targets protein phosphatase 2A to mitochondria and modulates apoptosis. J Biol Chem 2003;278:24976–24985.
Doi A, Park IH, Wen B, Murakami P, Aryee MJ, Irizarry R, Herb B, Ladd-Acosta C, Rho J, Loewer S, Miller J, Schlaeger T, Daley GQ, Feinberg AP. Differential methylation of tissue- and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibroblasts. Nat Genet 2009;41:1350–1353.
Drewes G, Mandelkow EM, Baumann K, Goris J, Merlevede W, Mandelkow E. Dephosphorylation of tau protein and Alzheimer paired helical filaments by calcineurin and phosphatase-2A. FEBS Lett 1993;336:425–432.
Durr A. Autosomal dominant cerebellar ataxias: polyglutamine expansions and beyond. Lancet Neurol 2010;9:885–894.
Esteller M. Epigenetics in cancer. N Engl J Med 2008;358:1148–1159.
Feinberg AP. Phenotypic plasticity and the epigenetics of human disease. Nature 2007;447:433–440.
Gong CX, Grundke-Iqbal I, Iqbal K. Dephosphorylation of Alzheimer's disease abnormally phosphorylated tau by protein phosphatase-2A. Neuroscience 1994;61:765–772.
Gong CX, Shaikh S, Wang JZ, Zaidi T, Grundke-Iqbal I, Iqbal K. Phosphatase activity toward abnormally phosphorylated tau: decrease in Alzheimer disease brain. J Neurochem 1995;65:732–738.
Harding AE. Classification of the hereditary ataxias and paraplegias. Lancet 1983;1:1151–1155.
Hardy J. Amyloid, the presenilins and Alzheimer's disease. Trends Neurosci 1997;20:154–159.
Hernández F, Avila J. Tauopathies. Cell Mol Life Sci 2007;64:2219–2233.
Hogarth P, Lovrecic L, D Krainc. Sodium phenylbutyrate in Huntington's disease: a dose-finding study. Mov Disord 2007;22:1962–1964.
Holmes SE, O'Hearn EE, McInnis MG, Gorelick-Feldman DA, Kleiderlein JJ, Callahan C, Kwak NG, Ingersoll-Ashworth RG, Sherr M, Sumner AJ, Sharp AH, Ananth U, Seltzer WK, Boss MA, Vieria- Saecker AM, Epplen JT, Riess O, Ross CA, Margolis RL. Expansion of a novel CAG trinucleotide repeat in the 5' region of PPP2R2B is associated with SCA12. Nat Genet 1999;23:391–392.
Holmes SE, Hwang H, O'Hearn E, Antonarakis SE, Chip S, Strck S, Ross CA, Margolis RL. Genomic structure and splice variants of PPP2R2B the gene associated with spinocerebellar ataxia type 12 (SCA12). Abstract Am J Hum Genet 2002;71:973.
Holmes SE, O'Hearn E, Margolisa RL. Why is SCA12 different from other SCAs? Cytogenet Genome Res 2003;100:189–197.
Huertas D, Sendra R, Munoz P. Chromatin dynamics coupled to DNA repair. Epigenetics 2009;4:31–42.
Ingebritsen TS, Cohen P. The protein phosphatases involved in cellular regulation. 1. Classification and substrate specificities, Eur J Biochem 1983;132:255–261.
Janssens V, Goris J. Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signaling. Biochem J 2001;353:417–439.
Janssens V, Goris J, Van Hoof C. PP2A: the expected tumor suppressor. Curr Opin Genet Dev 2005;15:34–41.
Ji H, Ehrlich LI, Seita J, Murakami P, Doi A, Lindau P, Lee H, Aryee MJ, Irizarry RA, Kim K, Rossi DJ, Inlay MA, Serwold T, Karsunky H, Ho L, Daley GQ, Weissman IL, Feinberg AP. Comprehensive methylome map of lineage commitment from haematopoietic progenitors. Nature 2010;467:338–342.
Jones PA, Baylin SB. The epigenomics of cancer. Cell 2007;128:683–692.
Kacem S, Feil R. Chromatin mechanisms in genomic imprinting. Mamm Genome 2009;20:544–556.
Kontopoulos E, Parvin JD, Feany MB. Alpha-synuclein acts in the nucleus to inhibit histone acetylation and promote neurotoxicity. Hum Mol Genet 2006;15:3012–3023.
Luco RF, Pan Q, Tominaga K, Blencowe BJ, Pereira-Smith OM, Misteli T. Regulation of alternative splicing by histone modifications. Science 2010;327:996–1000.
Mayer RE, Hendrix P, Cron P, Mattheis R, Stone SR, Goris J, Merlevede W, Hofsteenge J, Hemmings BA. Structure of the 55-kDa regulatory subunit of protein phosphatase 2A: evidence for a neuronal specific isoforms. Biochemistry 1991;30:3589–3597.
Mumby M. PP2A: unveiling a reluctant tumor suppressor. Cell 2007;130:21–24.
Myung NH, Zhu X, Kruman II, Castellani RJ, Petersen RB, Siedlak SL, Perry G, Smith MA, Lee HG. Evidence of DNA damage in Alzheimer disease: phosphorylation of histone H2AX in astrocytes. Age 2008;30:209–215.
O'Hearn E, Holmes SE, Calvert PC, Ross CA, Margolis RL. SCA12: Tremor with cerebellar and cortical atrophy is associated with a CAG repeat expansion. Neurology 2001;56:299–303.
Parihar MS, Hemnani T. Alzheimer's disease pathogenesis and therapeutic interventions. J Clin Neurosci 2004;11:456–467.
Portela A, Esteller M. Epigenetic modifications and human disease. Nat Biotechnol 2010;28:1057–1068.
Qian W, Shi J, Yin X, Iqbal K, Grundke-Iqbal I, Gong CX, Liu F. PP2A regulates tau phosphorylation directly and also indirectly via activating GSK-3beta. J Alzheimers Dis 2010;19:1221–1229.
Rando OJ, Chang HY. Genome-wide views of chromatin structure. Annu Rev Biochem 2009;78:245–271.
Reik W, Lewis A. Co-evolution of X-chromosome inactivation and imprinting in mammals. Nat Rev Genet 2005;6:403–410.
Ryu H, Lee J, Hagerty SW, Soh BY, McAlpin SE, Cormier KA, Smith KM, Ferrante RJ. ESET/SETDB1 gene expression and histone H3 (K9) trimethylation in Huntington's disease. Proc Natl Acad Sci USA 2006;103:19176–19181.
Sadri-Vakili G, Bouzou B, Benn CL, Kim MO, Chawla P, Overland RP, Glajch KE, Xia E, Qiu Z, Hersch SM, Clark TW, Yohrling GJ, Cha JH. Histones associated with downregulated genes are hypo-acetylated in Huntington's disease models. Hum Mol Genet 2007;16:1293–1306.
Shi Y. Serine/threonine phosphatases: Mechanism through structure. Cell 2009;139:468–484.
Shioi J, Georgakopoulos A, Mehta P, Kouchi Z, Litterst CM, Baki L, Robakis NK. FAD mutants unable to increase neurotoxic A 42 suggest that mutation effects on neurodegeneration may be independent of effects on Abeta. J Neurochem 2007;101:674–681.
Srivastava AK, Choudhry S, Gopinath MS, Roy S, Tripathi M, Brahmachari SK, Jain S. Molecular and clinical correlation in five Indian families with spinocerebellar ataxia 12. Ann Neurol 2001;50:796–800.
Steffan JS, Bodai L, Pallos J, Poelman M, McCampbell A, Apostol BL, Kazantsev A, Schmidt E, Zhu YZ, Greenwald M, Kurokawa R, Housman DE, Jackson GR, Marsh JL, Thompson LM. Histone deacetylase inhibitors arrest polyglutamine-dependent neurodegeneration in Drosophila. Nature 2001;413:739–743.
Strack S, Zaucha JA, Ebner FF, Colbran RJ, Wadzinski BE. Brain protein phosphatase 2A: developmental regulation and distinct cellular and subcellular localization by B subunits. J Comp Neurol 1998;392:515–527.
Straussman R, Nejman D, Roberts D, Steinfeld I, Blum B, Benvenisty N, Simon I, Yakhini Z, Cedar H. Developmental programming of CpG island methylation profiles in the human genome. Nat Struct Mol Biol 2009;16:564–571.
Strittmatter WJ, Saunders AM, Schmechel D, Pericak-Vance M, Enghild J, Salvesen G S, Roses A D. Apolipoprotein E: high-avidity binding to beta-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease. Proc Natl Acad Sci USA 1993;90:1977–1981.
Takai D, Jones PA. The CpG island searcher: a new WWW resource. In Silico Biol 2003;3:235–240.
Teive HA. Spinocerebellar ataxias. Arq Neuropsiquiatr 2009;67:1133–1142.
Turner PR, O'Connor K, Tate WP, Abraham WC. Roles of amyloid precursor protein and its fragments in regulating neural activity, plasticity and memory. Prog Neurobiol 2003;70:1–32.
Urdinguio RG, Sanchez-Mut JV, Esteller M. Epigenetic mechanisms in neurological diseases: genes, syndromes, and therapies. Lancet Neurol 2009;8:1056–1072.
Virshup DM. Protein phosphatase 2A: a panoply of enzymes. Curr Opin Cell Biol 2000;12:180–185.
Wade PA. Methyl CpG binding proteins: coupling chromatin architecture to gene regulation. Oncogene 2001 20:3166–3173.
Wu YR, Lin HY, Chen CM, Gwinn-Hardy K, Ro LS, Wang YC, Li SH, Hwang JC, Fang K, Hsieh-Li HM, Li ML, Tung LC, Su MT, Lu KT, Lee-Chen GJ. Genetic testing in spinocerebellar ataxia in Taiwan: expansions of trinucleotide repeats in SCA8 and SCA17 are associated with typical Parkinson's disease. Clin Genet 2004;65:209–214.
Xu Y, Chen Y, Zhang P, Jeffrey PD, Shi Y. Structure of a protein phosphatase 2A holoenzyme: insights into B55-mediated Tau dephosphorylation. Mol Cell 2008;31:873–885.
Yamada M, Sato T, Tsuji S, Takahashi H. CAG repeat disorder models and human neuropathology: similarities and differences. Acta Neuropathol 2008;115:71–86.