臺灣博碩士論文加值系統

第三型脊髓小腦運動失調症(Spinocerebellar ataxia type 3;SCA3)和第七型脊髓小腦運動失調症(Spinocerebellar ataxia type 7;SCA7)皆為遺傳型顯性神經退化性疾病，神經病變的成因是由於CAG核酸序列分別在其基因中不正常的增長，造成蛋白產物第三型運動失調質(ataxin-3)及第七型運動失調質(ataxin-7)中帶有多麩胺酸序列(polyglutamine)所致。而此含有突變多麩胺酸序列的蛋白，被認為就是造成神經病變的元兇。在先前的實驗中發現突變第三型運動失調質及突變第七型運動失調質經由增加Bax表現，進而活化由粒線體主導之凋亡路徑引起神經元的細胞凋亡(apoptosis)。進一步探究突變第三型運動失調質及突變第七型運動失調質引起神經元死亡之病理機轉，將可用於發展治療SCA3及SCA7的方法。因此，在本實驗我們進一步探討突變第三型運動失調質及突變第七型運動失調質引起Bax表現增加的分子機轉。
許多的證據指出，p53異常活化在許多神經退化性疾病的神經元死亡中扮演重要的角色，包括阿茲海默症(Alzheimer’s disease)、帕金森氏症(Parkinson’s disease)和亨汀頓氏症(Huntington’s disease)等等。而p53導致神經細胞死亡的機制，被認為是藉由調節促細胞凋亡(pro-apoptotic)基因的表現例如Bax、Puma及Noxa等。因此我們在本實驗的目的，便是要探討突變第三型運動失調質及突變第七型運動失調質所引起的Bax表現增加和神經細胞死亡，是否藉由活化的p53所調控。
利用電泳遷移率變動分析(Electrophoretic mobility shift assay; EMSA)檢測轉錄因子p53對Bax啟動子(promoter)的結合能力，分析突變第三型運動失調質及突變第七型運動失調質是否使得p53的轉錄能力上升。電泳遷移率變動分析的結果顯示，在表現了突變第三型運動失調質及突變第七型運動失調質的離體細胞模式(in vitro cellular model)或活體動物實驗(in vivo animal model)，p53對Bax啟動子的結合能力皆有增加的現象； 即時反轉錄-聚合酶鏈反應(Real-time RT-PCR)的結果顯示，受p53調控之基因Puma，在表現了突變第三型運動失調質及突變第七型運動失調質的培養小腦神經細胞及SCA3基因轉殖鼠和SCA7基因轉殖鼠受影響之腦區，Puma mRNA表現量皆有增加的現象，但是p53 mRNA的表現量並無變化。
綜合我們的結果顯示，突變第三型運動失調質及突變第七型運動失調質是藉由活化p53來增加Bax和Puma的表現而導致神經細胞的死亡，而由於p53的表現量並無變化，推測p53轉錄能力增加可能是透過轉譯後修飾作用造成。未來在SCA3及SCA7之病理機轉應該更進一步的研究，以期能發展出可行的治療策略。

Spinocerebellar ataxia type 3 (SCA3) and spinocerebellar ataxia type 7 (SCA7) are inherited autosomal dominant neurodegenerative disorders caused by abnormal expansion of CAG repeats coding for polyglutamine tract within protein called ataxin-3 and ataxin-7 respectively. The pathological basis of polyglutamine diseases is believed to be the dominant and neurotoxic property acquired by mutant polyglutamine-expanded proteins. Our previous studies demonstrated that mutant polyglutamine ataxin-3-Q79 and ataxin-7-Q75 activated mitochondrial apoptotic pathway and induced neuronal death by upregulating Bax expression. For the development of effective therapy for SCA3 and SCA7, better understanding the molecular basis of mutant ataxin-3- and ataxin-7-induced neurodegeneration is necessary. According to this reason, we investigated molecular mechanisms underlying polyglutamine-expanded ataxin-3-Q79- or ataxin-7-Q75-induced upregulation of Bax expression.
Several lines of evidence indicate that aberrant activation of p53 plays an important role in apoptotic neuronal death observed in several neurodegenerative disorders including stroke, Alzheimer’s disease, Huntington’s disease, Parkinson’s disease and amyotrophic lateral sclerosis. It is generally believed that p53 promotes neuronal apoptosis by enhancing the transcription of pro-apoptotic genes such as Bax and BH3-only Bcl-2 family members Noxa and Puma. In the present study, we tested the hypothesis that p53 activation is involved in mutant polyglutamine-expanded ataxin-3- and ataxin-7-induced upregulation of Bax expression and resulting apoptotic neuronal death.
Electrophoretic mobility shift assay (EMSA) using radiolabeled consensus p53 binding site of Bax promoter was performed to test the possibility that mutant ataxin-3-Q79 or ataxin-7-Q75 increases the p53 transcriptional activity. EMSA assays indicate that DNA-binding activity of p53 was increased in neurons expression mutant ataxin-3-Q79 or ataxin-7-Q75 both in vitro and in vivo. The mRNA level of Puma, a p53-inducible gene, was also upregulated in cultured cerebellar neurons expressing polyglutamine-expanded ataxin-3 or ataxin-7 and affected brain areas of ataxin-3-Q79 transgenic mice and ataxin-7-Q75 transgenic mice. However, mutant polyglutamine ataxin-3 and ataxin-7 fail to alter p53 mRNA expression both in vitro and in vivo.
In summary, our results indicate that polyglutamine-expanded ataxin-3 and ataxin-7 induces apoptotic neuronal death by increasing Bax and Puma expressions via activating p53. Mutant ataxin-3-Q79 or ataxin-7-Q75 fails to alter the expression level of p53, suggesting that mutant ataxin-3-Q79 or ataxin-7-Q75 enhances p53 transcriptional activity by post-translational modification of p53. Future investigation of molecular mechanisms underlying ataxin-3-Q79 and ataxin-7-Q75-induced neuronal death should shed a light on the pathogenic mechanism of SCA3 and SCA7 and lead to a development of possible therapeutic strategies.

Abstract (Chinese) v
Abstract (English) vii
Abbreviations ix
Contents xi
I. Introduction 1
II. Specific aims 8
III. Materials and methods 9
3.1 Preparation of primary cultured neurons 9
3.2 Preparation of recombinant adenoviruses 9
3.3 Generation of transgenic mice 10
3.4 Nuclear and cytosolic extracts 11
3.5 Electrophoretic mobility shift assay of p53 DNA binding activity 12
3.6 Real-time quantitative RT-PCR assay 12
3.7 Western blotting analysis of p53 13
3.8 Statistics 14
IV. Results 15
4.1 Polyglutamine-expanded ataxin-3-Q79 or ataxin-7-Q75 enhance
DNA binding activity of p53 in cultured cerebellar neurons 15
4.2 Polyglutamine-expanded ataxin-3-Q79 or ataxin-7-Q75 increase DNA-binding activity of p53 in vivo 16
4.3 Polyglutamine-expanded ataxin-3-Q79 or ataxin-7-Q75 upregulates Puma mRNA expression in cultured cerebellar neurons 17
4.4 Polyglutamine-expanded ataxin-3-Q79 or ataxin-7-Q75 upregulates Puma mRNA expression in vivo 18
4.5 Polyglutamine-expanded ataxin-3-Q79 or ataxin-7-Q75 fails to alter p53 mRNA expression 18
V. Disscussion 20
VI. References 26
VII. Figures 34

Adams JM, Cory S (2007) The Bcl-2 apoptotic swich in cancer development and therapy. Oncogene 26, 1324-1337.
Bae BI, Xu H, Igarashi S, Fujimoto M, Agrawai N, Taya Y, Hayward SD, Moran T H, Montell C, Ross CA, Snyder SH, Sawa A (2005) p53 mediates cellular dysfunction and behavioral abnormalities in Huntington’s disease. Neuron 47, 29-41.
Borchelt DR, Davis J, Fischer M, Lee MK, Slunt HH, Ratovitsky T, Regard J, Copeland NG, Jenkins NA, Sisodia SS, Price DL (1996) A vector for expressing foreign genes in the brains of hearts of transgenic mice. Genet. Anal. 13, 159-163.
Chen L, Willis SN, Wei A, Smith BJ, Fletcher JI, Hinds MG, Colman PM, Day CL, Adams JM, Huang DCS (2005). Differential targeting of pro-survival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function. Mol. Cell 17, 393-403.
Chipuk JE, Green DR (2006) Dissecting p53-dependent apoptosis. Cell Death Differ. 13, 94-1002.
Chou AH, Yeh TH, Kuo YL, Kao YC, Jou MJ, Hsu CY, Tsai SR, Kakizuka A, Wang HL (2006) Polyglutamine-expanded ataxin-3 activates mitochondrial apoptotic pathway by upregulating Bax and downregulating Bcl-xL. Neurobiol. Dis. 21, 335-345.
Culmsee C, Mattson MP (2005) p53 in neuronal apoptosis. Biochem. Biophys. Res. Commun. 331, 761-777.
David G, Abbas N, Stevanin G, Durr A, Yvert G, Cancel G, Weber C, Imbert G, Saudou F, Antoniou E, Drabkin H, Gemmill R, Giunti P, Benomar A, Wood N, Ruberg M, Agid Y, Mandel JL, Brice A (1997) Cloning of the SCA7 gene reveals a highly unstable CAG repeat expansion. Nature Genet. 17, 65-70.
David G, Durr A, Stevanin G, Cancel G, Abbas N, Yvert G, Benomar A, Belal S, Lebre AS, Abada-Bendib M, Grid D, Holmberg M, Yahyaoui M, Hentati F, Chkill T, Agid Y, Brice A (1998) Molecular and clinical correlations in autosomal dominant cerebellar ataxia with progressive macular dystrophy. Hum. Mol. Genet. 7, 165-170.
Duenas A.M., Goold R., Giunti P (2006) Molecular pathogenesis of spinocerebellar ataxias. Brain 129, 1357-1370.
Durr A, Stevanin G, Cancel G, Duyckaerts C, Abbas N, Didierjean O, Chneiweiss H, Benomar A, Lyon-Caen O, Julien J, Serdaru M, Penet C, Agid Y, Brice A (1996) Spinocerebellar ataxia 3 and Machado-Joseph disease: clinical, molecular, and neuropathological features. Ann. Neurol. 39, 490-499.
Everett CM, Wood NW (2004) Trinucleotide repeats and neurodegenerative disease. Brain 127, 2385-2405.
Fridman JS, Lowe SW (2003) Control of apoptosis by p53. Oncogene 22, 9030-9040.
Gonzalez de Aguilar JL, Gordon JW, Rene F, de Tapia M, Lutz-Bucher B, Gaiddon C, Loeffler JP (2000) Alteration of the Bcl-x/Bax ratio in a transgenic mouse model of amyotrophic lateral sclerosis: evidence for the implication of the p53 signaling pathway. Neurobiol. Dis. 7, 406-415.
Gusella JF, MacDonald M (2000) Molecular genetics: unmasking polyglutamine triggers in neurodegenerative disease. Nat. Rev. 1, 109-115.
He TC, Zhou SB, Costa LT, Yu J, Kinzler KW, Vogelstein B (1998) A simplified system for generating recombinant adenoviruses. Proc. Natl. Acad. Sci. USA 95, 2509-2514.
Hofseth LJ, Hussain SP, Harris CC (2004) p53: 25 years after its discovery. Trends in Pharmacol. Sci. 25, 177-181.
Kawaguchi Y, Okamoto T, Taniwaki M, Aizawa M, Inoue M, Katayama S, Kawakami H, Nakamura S, Nishimura M, Akiguchi I, Kimura J, Narumiya S, Kakizuka A (1994) CAG expansions in a novel gene for Machado-Joseph disease at chromosome 14q32.1. Nature Genet. 8, 221-228.
Kaytor MD, Duvick LA, Skinner PJ, Koob MD, Ranum LPW, Orr HT (1999) Nuclear localization of the spinocerebellar ataxia type 7 protein, ataxin-7. Hum. Molec. Genet. 8, 1657-1664.
Kobayashi T, Kakizuka A (2003) Molecular analysis of Machado-Joseph disease. Cytogenet Genome Res. 100, 261-275.
Koulich E, Nguyen T, Johnson K, Giardina CA,D’Mello SR (2001) NF-B is involved in the survival of cerebellar granule neurons: association of IB phosphorylation with cell survival. J. Neurochem. 76, 1188-1198.
Lavin MF, Gueven N (2006) The complexity of p53 stabilixation and activation. Cell Death Differ. 13, 941-950.
Lebre AS, Brice A (2003) Spinocerebellar ataxia-7. Cytogenet Genome Res. 100, 154-163.
Lee JH, Kim KT (2004) Induction of cyclin-dependent kinase 5 and its activator p35 through the extracellular-signal-regulated kinase and protein kinase A pathways during retinoic-acid mediated neuronal differentiation in human neuroblastoma SK-NBE(2)C cells. J. Neurochem. 91, 634-647.
Lee JH, Kim HS, Lee SJ, Kim KT (2007) Stabilization and activation of p53 induced by Cdk5 contributes to neuronal cell death. J. Cell Sci. 120, 2259-2271.
Lindenberg KS, Yvert G, Müller K, Landwehrmeyer GB (2000) Expression analysis of ataxin-7 mRNA and protein in human brain: Evidence for a widespread distributionand focal protein accumulation. Brain Pathol. 10, 385-394.
Martin JJ, van Regemorter N, Krols L, Brucher JM, de Barsy T, Szliwowski H, Evrard P, Ceuterick C, Tassuignon MT, Smet-Dieleman H, Van Broeckhoven C (1994) On a autosomal dominant form of retinal-cerebellar degeneration: an autopsy study of five patients in one family. Acta. Neuoropathol. 88, 277-286.
Miyasita T, Reed JC (1995) Tumor suppressor p53 is a direct transcriprional activator of the human bax gene. Cell 80, 293-299.
Morrison RS, Konoshita Y, Johnson MD, Guo W, Garden GA (2003) p53-dependent cell death signaling in neurons. Neruochem. Res. 28, 15-27
Nakamura K, Jeong SY, Uchihara T, Anno M, Nagashima K, Nagashima T, Ikeda S, Tsuji S, Kanazawa I (2001) SCA17, a novel autosomal dominant cerebellar ataxia caused by an expanded polyglutamine in TATA-binding protein. Hum. Mol. Genet. 14, 1441-1448.
Nakano K and Vousden KH (2001) PUMA, a novel proapoptotic gene, is induced by p53. Mol. Cell 7, 683-694.
Nguyen MD, Lariviere RC, Julien JP (2001) Deregulation of Cdk5 in a mouse model of ALS: toxicity alleviated by perikaryal neurofilament inclusions. Neuron 30, 135-147.
Oda E, Ohki R, Murasawa H, Nemoto J, Shibue T, Yamashita T, Tokino T, Taniguchi T, Tanaka N (2000) Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis. Science 288, 1053-1058
Okazawa H (2003) Polyglutamine diseases: a transcription disorder ? Cell. Mol. Life Sci. 60, 1427-1439.
Patrick GN, Zukerberg L, Nikolic M, de la Monte S, Dikkes P, Tsai LH (1999) Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration. Nature 402, 615-622.
Paulson HL, Das SS, Crino PB, Perez MK, Patel SC, Gotsdiner D, Fischbeck KH, Pittman RN (1997a) Machado-Joseph disease gene product is a cytoplasmic protein widely expressed in brain. Ann. Neurol. 41, 453- 462.
Paulson HL (2000) Toward an understanding of polyglutamine neurodegeneration. Brain.Pathol. 10, 293-299.
Paulson HL, Bonini NM, Roth KA (2000) Polyglutamine disease and neuronal cell death. Pro. Natl. Acad. Sci. USA 97, 12957-12958.
Ramalho RM, Ribeiro PS, Sola’ S, Castro RE, Steer CJ, Rodrigues CMP (2004) Inhibition of the E2F-1/p53/Bax pathway by tauroursodeoxycholic acid in amyloid -peptide-induced apoptosis in PC12 cells. J. Neurochem. 90, 567-575.
Rosenberg RN (1992) Machado-Joseph disease: An autosomal dominant system degeneration. Mov. Dis. 3, 193-203.
Schilling G, Becher MW, Sharp AH, Jinnah HA, Duan K, Kotzuk JA, Slunt HH, Ratovitski T, Cooper JK, Jenkins NA, Copeland NG, Price DL, Ross CA, Borchelt DR (1999) Intranuclear inclusions and neuritic aggregates in transgenic mice expressing a mutant N-terminal fragment of huntingtin. Hum Mol Genet. 8, 397-407.
Shelton BS, Johnson GVW (2004) Cyclin-dependent kinase-5 in neurodegeneration. J. Neurochem. 88, 1313-1326.
Shibue T, Suxuki S, Okamoto H, Yoshida H, Yusuke O, Takaoka A, Taniguchi T (2006) Differential contribution of Puma and Noxa in dual regulation of p53-mediated apoptotic pathways. EMBO J. 25, 4952-4962.
Smith DS, Greer PL, Tsai LH (2001) Cdk5 on the brain. Cell Growth Differ. 12, 277-283.
Sugars KL and Rubinsztein DC (2003) Transcriptional abnormalities in Huntington’s disease. Trends in Genet. 19, 233-238.
Sykes SM, Mellert HS, Holbert MA, Li K, Marmorstein R, Lane WS, McMahon SB (2006) Acetylation of the p53 DNA-binding domain regulates apoptosis induction. Mol. Cell 24, 841-851.
Tait D, Riccio M, Sittler A, Scherzinger E, Santi S, Ognibene A, Maraldi NM, Lehrach H, Wanker EE (1998) Ataxin-3 is transported into the nucleus and associates with the nuclear matrix. Hum. Molec. Genet. 7, 99-997.
Takiyama Y, Oyanagi S, Kawashima S, Sakamoto H, Saito K, Yoshida M, Tsuji S, Mizuno Y and Nishizawa M (1994) A clinical and pathologic study of a large Japanese family with Machado-Joseph disease tightly linked to the DNA markers on chromosome 14q. Neurology 44, 1302-1308.
Tanemura K, Murayama M, Akagi T, Hashikawa T, Tominaga T, Ichikawa M, Yamaguchi H, Takashima A (2002) Neurodegeneration with tau accumulation in a transgenic mouse expressing V337M human tau. J. Neurosci. 22, 133-141.
Villunger A, Michalak EM, Coultas L, Mullauer F, Bock G, Ausserlechner MJ, Adams JM, Strasser A (2003) p53- and drug-induced apoptotic responses mediated by BH3-only proteins puma and noxa. Science 302, 1036-1038.
Wang HL, Chang WT, Hsu CY, Huang PC, Chow YW, Li A (2002) Identification of two C-terminal amino acids, Ser355 and Thr357, required for short-term homologous desensitization of -opioid receptors. Biochem.Pharmacol. 64, 257-266.
Wang HL, Yeh TH, Chou AH, Kuo YL, Luo LJ, He CY, Huang PC, Li AH (2006) Polyglutamine-expanded ataxin-7 activates mitochondrial apoptotic pathway of cerebellar neurons by upregulating Bax and downregulating Bcl-xL. Cell. Signal. 18, 541-552.
Wang HL, He CY, Chou AH, Yeh TH, Chen YL, Li AH (2007) Polyglutamine-expanded ataxin-7 decreases nuclear translocation of NF-B activity by inhibiting proteasome activity of cerebellar neurons. Cell. signal. 19, 573-581.
Ward MW, Kogel D, Prehn JH (2004) Neuronal apoptosis: BH3-only proteins the real killers? J. Bioenerg. Biomembr. 36, 295-298.
Willis SN and Adams JM (2005) Life in the balance: how BH3-only proteins induce apoptosis. Curr. Opin. Cell Biol. 17, 617-625.
Xiang H, Kinoshita Y, Knudson CM, Korsmeyer SJ, Schwartzkroin PA, Morrison RS (1998) Bax involvement in p53-mediated neuronal cell death. J. Neurosci. 18, 1363- 1373.
Yeh TH, Hwang HM, Chen JJ, Wu T, Li A, Wang HL (2005) Glutamate transporter function of rat hippocampal astrocytes is impaired following the global ischemia. Neurobiol. Dis. 18, 476-483.
Yu J, Zhang L (2005) The transcriptional targets of p53 in apoptosis control. Biochem. Biophys. Res. Commun. 331, 851-858.
Zamzami N, Kroemer G (2005) p53 in apoptosis control: an introduction. Biochem Biophys Res Commun. 331, 685-687.
Zhang J, Krishnamurthy PK, Johnson GVW (2002) Cdk5 phosphorylates p53 and regulates its activity. J. Neurochem. 81, 307-313.
Zoghbi HY, Orr HT (1999) Polyglutamine diseases: protein cleavage and aggregation. Curr Opin. Neurobiol. 9, 566-570.
Zoghbi HY, Orr HT (2000) Glutamine repeats and neurodegeneration. Annu. Rev. Neurosci. 23, 213-247.