目前的研究指出脊髓小腦運動失調症12型(Spinocerebellar ataxia type 12, SCA12) 可能是PPP2R2B基因的5’端未轉譯區域的CAG三核苷酸擴充所致。PPP2R2B基因轉譯出具有腦特異性之Bβ調控次單元，這個Bβ調控次單元為蛋白去磷酸酶2A(PP2A)的其中一單元。由於5’端未轉譯區的不正常擴增，可能使得基因PPP2R2B產生選擇性剪切(alternative splicing)，而轉譯出其中的一個Bβ調節次單元Bβ2。過去的文獻提出Bβ2-PP2A會有粒腺體專一性，使Bβ2-PP2A停留在粒腺體外膜造成類神經細胞株PC-12凋亡。在細胞模式上，我們利用脂質體攜帶Bβ2基因送入神經母細胞瘤細胞株SH-SY5Y，並處理視網酸(retinoic acid)使其分化為似神經的成熟細胞來進行觀察過量Bβ2表現是否位於粒腺體上，及Bβ2過度表現對細胞所造成的氧化壓力。在活細胞系統的長時間連續性觀察中，我們可以發現過度表現Bβ2的SH-SY5Y細胞有較對照組不正常的細胞型態。在了解到Bβ2在細胞中所參與的角色，期能在未來提供一個藥物篩選的平台。

Spinocerebellar ataxia type 12 (SCA12) is associated with an expansion of a CAG repeats in the 5’region of the gene PPP2R2B, which encodes a brain-specific regulatory subunit (Bβ) of the protein phosphatase 2A (PP2A). One of the splice variants of PPP2R2B, Bβ2, has been reported to target PP2A to mitochondria to promote apoptosis in PC12 cells. In our cell model, we used liposome based transient transfection overexpressing Bβ2. The cellular localization of Bβ2 and oxidative stress stimulation by Bβ2 was characterized in retinoic acid differentiated SH-SY5Y neuroblastoma cell line. These tranfected SH-SY5Y cells were characterized on a time-lapsed live image recorded system. Further analysis using Western blot, and immunocytochemistry will be conducted to study the molecular significance. Hoping this system could provide a neurodegenerative platforms for cell model based pathological analysis and potential compound screening.

Alexa A, Schmidt G, Tompa P, Ogueta S, Vazquez J, Kulcsar P, Kovacs J, Dombradi V, Friedrich P (2002) The phosphorylation state of threonine-220, a uniquely phosphatase-sensitive protein kinase A site in microtubule-associated protein MAP2c, regulates microtubule binding and stability. Biochemistry 41:12427-12435.
Alves S, Regulier E, Nascimento-Ferreira I, Hassig R, Dufour N, Koeppen A, Carvalho AL, Simoes S, de Lima MC, Brouillet E, Gould VC, Deglon N, de Almeida LP (2008) Striatal and nigral pathology in a lentiviral rat model of Machado-Joseph disease. Hum Mol Genet 17:2071-2083.
Ashe PC, Berry MD (2003) Apoptotic signaling cascades. Prog Neuropsychopharmacol Biol Psychiatry 27:199-214.
Bohlen M, Cameron A, Metten P, Crabbe JC, Wahlsten D (2009) Calibration of rotational acceleration for the rotarod test of rodent motor coordination. J Neurosci Methods 178:10-14.
Bosboom JL, Stoffers D, Wolters E (2004) Cognitive dysfunction and dementia in Parkinson's disease. J Neural Transm 111:1303-1315.
Brunet A, Datta SR, Greenberg ME (2001) Transcription-dependent and -independent control of neuronal survival by the PI3K-Akt signaling pathway. Curr Opin Neurobiol 11:297-305.
Brusco A, Gellera C, Cagnoli C, Saluto A, Castucci A, Michielotto C, Fetoni V, Mariotti C, Migone N, Di Donato S, Taroni F (2004) Molecular genetics of hereditary spinocerebellar ataxia: mutation analysis of spinocerebellar ataxia genes and CAG/CTG repeat expansion detection in 225 Italian families. Arch Neurol 61:727-733.
Cagnoli C, Mariotti C, Taroni F, Seri M, Brussino A, Michielotto C, Grisoli M, Di Bella D, Migone N, Gellera C, Di Donato S, Brusco A (2006) SCA28, a novel form of autosomal dominant cerebellar ataxia on chromosome 18p11.22-q11.2. Brain 129:235-242.
Chang L, Karin M (2001) Mammalian MAP kinase signalling cascades. Nature 410:37-40.
Dagda RK, Zaucha JA, Wadzinski BE, Strack S (2003) A developmentally regulated, neuron-specific splice variant of the variable subunit Bbeta targets protein phosphatase 2A to mitochondria and modulates apoptosis. J Biol Chem 278:24976-24985.
Dagda RK, Barwacz CA, Cribbs JT, Strack S (2005) Unfolding-resistant translocase targeting: a novel mechanism for outer mitochondrial membrane localization exemplified by the Bbeta2 regulatory subunit of protein phosphatase 2A. J Biol Chem 280:27375-27382.
Datta SR, Katsov A, Hu L, Petros A, Fesik SW, Yaffe MB, Greenberg ME (2000) 14-3-3 proteins and survival kinases cooperate to inactivate BAD by BH3 domain phosphorylation. Mol Cell 6:41-51.
Duenas AM, Goold R, Giunti P (2006) Molecular pathogenesis of spinocerebellar ataxias. Brain 129:1357-1370.
Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, Naldini L (1998) A third-generation lentivirus vector with a conditional packaging system. J Virol 72:8463-8471.
Farber E (1994) Programmed cell death: necrosis versus apoptosis. Mod Pathol 7:605-609.
Gutteridge JM, Halliwell B (2000) Free radicals and antioxidants in the year 2000. A historical look to the future. Ann N Y Acad Sci 899:136-147.
Halliwell B (2006) Oxidative stress and neurodegeneration: where are we now? J Neurochem 97:1634-1658.
Harper SJ, LoGrasso P (2001) Signalling for survival and death in neurones: the role of stress-activated kinases, JNK and p38. Cell Signal 13:299-310.
Henze K, Martin W (2003) Evolutionary biology: essence of mitochondria. Nature 426:127-128.
Herzig S, Neumann J (2000) Effects of serine/threonine protein phosphatases on ion channels in excitable membranes. Physiol Rev 80:173-210.
Holmes SE, Hearn EO, Ross CA, Margolis RL (2001) SCA12: an unusual mutation leads to an unusual spinocerebellar ataxia. Brain Res Bull 56:397-403.
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 (1999) Expansion of a novel CAG trinucleotide repeat in the 5' region of PPP2R2B is associated with SCA12. Nat Genet 23:391-392.
Ishikawa K, Toru S, Tsunemi T, Li M, Kobayashi K, Yokota T, Amino T, Owada K, Fujigasaki H, Sakamoto M, Tomimitsu H, Takashima M, Kumagai J, Noguchi Y, Kawashima Y, Ohkoshi N, Ishida G, Gomyoda M, Yoshida M, Hashizume Y, Saito Y, Murayama S, Yamanouchi H, Mizutani T, Kondo I, Toda T, Mizusawa H (2005) An autosomal dominant cerebellar ataxia linked to chromosome 16q22.1 is associated with a single-nucleotide substitution in the 5' untranslated region of the gene encoding a protein with spectrin repeat and Rho guanine-nucleotide exchange-factor domains. Am J Hum Genet 77:280-296.
Janssens V, Goris J (2001) Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem J 353:417-439.
Jin Z, El-Deiry WS (2005) Overview of cell death signaling pathways. Cancer Biol Ther 4:139-163.
Kanninen K, Heikkinen R, Malm T, Rolova T, Kuhmonen S, Leinonen H, Yla-Herttuala S, Tanila H, Levonen AL, Koistinaho M, Koistinaho J (2009) Intrahippocampal injection of a lentiviral vector expressing Nrf2 improves spatial learning in a mouse model of Alzheimer's disease. Proc Natl Acad Sci U S A 106:16505-16510.
Knight MA, Gardner RJ, Bahlo M, Matsuura T, Dixon JA, Forrest SM, Storey E (2004) Dominantly inherited ataxia and dysphonia with dentate calcification: spinocerebellar ataxia type 20. Brain 127:1172-1181.
Kyriakis JM, Avruch J (2001) Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev 81:807-869.
Martindale JL, Holbrook NJ (2002) Cellular response to oxidative stress: signaling for suicide and survival. J Cell Physiol 192:1-15.
McBride HM, Neuspiel M, Wasiak S (2006) Mitochondria: more than just a powerhouse. Curr Biol 16:R551-560.
Merrick SE, Trojanowski JQ, Lee VM (1997) Selective destruction of stable microtubules and axons by inhibitors of protein serine/threonine phosphatases in cultured human neurons. J Neurosci 17:5726-5737.
Moreno CS, Park S, Nelson K, Ashby D, Hubalek F, Lane WS, Pallas DC (2000) WD40 repeat proteins striatin and S/G(2) nuclear autoantigen are members of a novel family of calmodulin-binding proteins that associate with protein phosphatase 2A. J Biol Chem 275:5257-5263.
Ouyang M, Shen X (2006) Critical role of ASK1 in the 6-hydroxydopamine-induced apoptosis in human neuroblastoma SH-SY5Y cells. J Neurochem 97:234-244.
Paulson HL (2009) The spinocerebellar ataxias. J Neuroophthalmol 29:227-237.
Pittman RN, Wang S, DiBenedetto AJ, Mills JC (1993) A system for characterizing cellular and molecular events in programmed neuronal cell death. J Neurosci 13:3669-3680.
Ruediger R, Pham HT, Walter G (2001) Alterations in protein phosphatase 2A subunit interaction in human carcinomas of the lung and colon with mutations in the A beta subunit gene. Oncogene 20:1892-1899.
Ruvolo PP, Deng X, Ito T, Carr BK, May WS (1999) Ceramide induces Bcl2 dephosphorylation via a mechanism involving mitochondrial PP2A. J Biol Chem 274:20296-20300.
Saito T, Shima H, Osawa Y, Nagao M, Hemmings BA, Kishimoto T, Hisanaga S (1995) Neurofilament-associated protein phosphatase 2A: its possible role in preserving neurofilaments in filamentous states. Biochemistry 34:7376-7384.
Sato N, Amino T, Kobayashi K, Asakawa S, Ishiguro T, Tsunemi T, Takahashi M, Matsuura T, Flanigan KM, Iwasaki S, Ishino F, Saito Y, Murayama S, Yoshida M, Hashizume Y, Takahashi Y, Tsuji S, Shimizu N, Toda T, Ishikawa K, Mizusawa H (2009) Spinocerebellar ataxia type 31 is associated with "inserted" penta-nucleotide repeats containing (TGGAA)n. Am J Hum Genet 85:544-557.
Scanlon JM, Reynolds IJ (1998) Effects of oxidants and glutamate receptor activation on mitochondrial membrane potential in rat forebrain neurons. J Neurochem 71:2392-2400.
Schols L, Bauer P, Schmidt T, Schulte T, Riess O (2004) Autosomal dominant cerebellar ataxias: clinical features, genetics, and pathogenesis. Lancet Neurol 3:291-304.
Sontag E, Hladik C, Montgomery L, Luangpirom A, Mudrak I, Ogris E, White CL, 3rd (2004a) Downregulation of protein phosphatase 2A carboxyl methylation and methyltransferase may contribute to Alzheimer disease pathogenesis. J Neuropathol Exp Neurol 63:1080-1091.
Sontag E, Luangpirom A, Hladik C, Mudrak I, Ogris E, Speciale S, White CL, 3rd (2004b) Altered expression levels of the protein phosphatase 2A ABalphaC enzyme are associated with Alzheimer disease pathology. J Neuropathol Exp Neurol 63:287-301.
Sontag E, Nunbhakdi-Craig V, Lee G, Brandt R, Kamibayashi C, Kuret J, White CL, 3rd, Mumby MC, Bloom GS (1999) Molecular interactions among protein phosphatase 2A, tau, and microtubules. Implications for the regulation of tau phosphorylation and the development of tauopathies. J Biol Chem 274:25490-25498.
Storey E, Bahlo M, Fahey M, Sisson O, Lueck CJ, Gardner RJ (2009) A new dominantly inherited pure cerebellar ataxia, SCA 30. J Neurol Neurosurg Psychiatry 80:408-411.
Strack S, Cribbs JT, Gomez L (2004) Critical role for protein phosphatase 2A heterotrimers in mammalian cell survival. J Biol Chem 279:47732-47739.
Strack S, Westphal RS, Colbran RJ, Ebner FF, Wadzinski BE (1997) Protein serine/threonine phosphatase 1 and 2A associate with and dephosphorylate neurofilaments. Brain Res Mol Brain Res 49:15-28.
Strack S, Zaucha JA, Ebner FF, Colbran RJ, Wadzinski BE (1998) Brain protein phosphatase 2A: developmental regulation and distinct cellular and subcellular localization by B subunits. J Comp Neurol 392:515-527.
Takagi Y, Futamura M, Yamaguchi K, Aoki S, Takahashi T, Saji S (2000) Alterations of the PPP2R1B gene located at 11q23 in human colorectal cancers. Gut 47:268-271.
Taroni F, DiDonato S (2004) Pathways to motor incoordination: the inherited ataxias. Nat Rev Neurosci 5:641-655.
Thannickal VJ, Fanburg BL (2000) Reactive oxygen species in cell signaling. Am J Physiol Lung Cell Mol Physiol 279:L1005-1028.
Torashima T, Koyama C, Iizuka A, Mitsumura K, Takayama K, Yanagi S, Oue M, Yamaguchi H, Hirai H (2008) Lentivector-mediated rescue from cerebellar ataxia in a mouse model of spinocerebellar ataxia. EMBO Rep 9:393-399.
Uo T, Kinoshita Y, Morrison RS (2005) Neurons exclusively express N-Bak, a BH3 domain-only Bak isoform that promotes neuronal apoptosis. J Biol Chem 280:9065-9073.
Van Kanegan MJ, Adams DG, Wadzinski BE, Strack S (2005) Distinct protein phosphatase 2A heterotrimers modulate growth factor signaling to extracellular signal-regulated kinases and Akt. J Biol Chem 280:36029-36036.
Walter G, Ferre F, Espiritu O, Carbone-Wiley A (1989) Molecular cloning and sequence of cDNA encoding polyoma medium tumor antigen-associated 61-kDa protein. Proc Natl Acad Sci U S A 86:8669-8672.
Wanisch K, Yanez-Munoz RJ (2009) Integration-deficient lentiviral vectors: a slow coming of age. Mol Ther 17:1316-1332.
Weeber EJ, Jiang YH, Elgersma Y, Varga AW, Carrasquillo Y, Brown SE, Christian JM, Mirnikjoo B, Silva A, Beaudet AL, Sweatt JD (2003) Derangements of hippocampal calcium/calmodulin-dependent protein kinase II in a mouse model for Angelman mental retardation syndrome. J Neurosci 23:2634-2644.
Yang SH, Cheng PH, Banta H, Piotrowska-Nitsche K, Yang JJ, Cheng EC, Snyder B, Larkin K, Liu J, Orkin J, Fang ZH, Smith Y, Bachevalier J, Zola SM, Li SH, Li XJ, Chan AW (2008) Towards a transgenic model of Huntington's disease in a non-human primate. Nature 453:921-924.