以SCA3誘導細胞模式進行潛力新藥之篩選

Abstract

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。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.