以SCA3誘導細胞模式進行潛力新藥之篩選

Screening Novel Potential Therapeutic Drugs for Spinocerebellar Ataxia Type 3 through Inducible Cell system

陳星蓉(Hsing-Jung Chen)

指導教授 ： 謝秀梅

國立臺灣師範大學/理學院/生命科學研究所/碩士(2011年)

摘要

小腦脊髓運動失調症第三型(spinocerebellar ataxia type 3; SCA3)，亦稱Machado–Joseph disease (MJD)，為小腦脊髓運動失調症眾多亞型中最常見的一型，屬於晚發性自體顯性遺傳的神經退化性疾病。主要是因為座落於14q24.3-q32的MJD1基因發生CAG三核苷酸過度擴增的現象，這些擴增的CAG序列會轉譯出含多麩醯胺酸(polyglutamine; polyQ)序列的蛋白質產物ataxin3 (AT3)，突變的AT3會在細胞內聚集，透過蛋白質水解機制切割後，在細胞核內形成包含體(nuclear inclusions; NIs)產生細胞毒性使得細胞死亡。目前SCA3實際的致病機轉尚未明瞭，為了解AT3與SCA3病程中的致病機制，我們建立AT3誘導表現的PC12細胞模式。我們發現75Q細胞在氧化壓力以及蛋白質酶抑制劑處理過後會比27Q細胞更容易產生蛋白質聚集在細胞核以及細胞和周圍的現象，對於壓力藥物的耐受性也比27Q細胞要低許多。因此我們藉由建立的SCA3細胞模式做一系列新穎組蛋白去乙醯酶抑制劑(histone deacetylase inhibitors; HDACi)藥物篩選，結果發現某些抑制劑藥物確實能保護SCA3細胞，提升細胞存活率以及神經細胞分支生長，降低氧化壓力以及蛋白質聚集的現象，並增加組蛋白(histone)乙醯化及活化許多具神經保護性的訊息傳遞路徑例如Hsp27、ERK、 MnSOD 和NF-κB等。因此，組蛋白去乙醯酶抑制劑藥物或許可成為治療SCA3的良好候選療法。

關鍵字

第三型脊髓小腦運動共濟失調症 ； 去乙醯化抑制劑 ； 氧化壓力 ； 神經退化性疾病

並列摘要

Spinocerebellar ataxia type 3 (SCA3), also called Machado–Joseph disease (MJD), is an autosomal dominant neurodegenerative disease results from expanded CAG repeat of MJD1 gene. The CAG repeat expansion encodes polyglutamine (polyQ) stretch in mutant ataxin-3 protein and causes protein cleaved, insoluble, accumulated and aggregated in the neurons. These inclusions further elevate cellular oxidative stress and lead to cell death. We established inducible cell system expressing human full length ataxin-3 containing 27Q or 75Q in PC12 cells. Cells with 75Q ataxin-3 showed lower viability and more nuclear/peri-nuclear aggregation in stress environments. We tested several novel HDAC inhibitor (HDACi) compounds in the inducible SCA3 cells. Our results show that some HDACi could elevate cell viability, reduce aggregation and promote neurite outgrowth. We also observed that HDACi could increase histone acetylation and activate neuroprotective proteins, such as Hsp27, ERK pathway regulators, MnSOD and NF-κB. These findings indicate that some novel HDACi compounds might be potential for SCA3 treatment.

並列關鍵字

Spinocerebellar Ataxia Type 3 ； polyQ ； HDACi ； oxidative stress

參考文獻

Aletta JM (1996) Phosphorylation of type III beta-tubulin PC12 cell neurites during NGF-induced process outgrowth. J Neurobiol 31:461-475.

Apostol BL, Illes K, Pallos J, Bodai L, Wu J, Strand A, Schweitzer ES, Olson JM, Kazantsev A, Marsh JL, Thompson LM (2006) Mutant huntingtin alters MAPK signaling pathways in PC12 and striatal cells: ERK1/2 protects against mutant huntingtin-associated toxicity. Hum Mol Genet 15:273-285.

Barbeau A, Roy M, Cunha L, de Vincente AN, Rosenberg RN, Nyhan WL, MacLeod PL, Chazot G, Langston LB, Dawson DM, et al. (1984) The natural history of Machado-Joseph disease. An analysis of 138 personally examined cases. Can J Neurol Sci 11:510-525.

Bence NF, Sampat RM, Kopito RR (2001) Impairment of the ubiquitin-proteasome system by protein aggregation. Science 292:1552-1555.

Benn CL, Butler R, Mariner L, Nixon J, Moffitt H, Mielcarek M, Woodman B, Bates GP (2009) Genetic knock-down of HDAC7 does not ameliorate disease pathogenesis in the R6/2 mouse model of Huntington's disease. PLoS One 4:e5747.