李桂楨高慈蓬2019-09-052011-8-42019-09-052010http://etds.lib.ntnu.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=id=%22GN0697430339%22.&%22.id.&http://rportal.lib.ntnu.edu.tw:80/handle/20.500.12235/104305PP2A為真核細胞內一種普遍的絲胺酸/酥胺酸去磷酸酶。PP2A全酶包含結構骨架A次單位、調節B次單位、催化C次單位。PPP2R2B (Bβ)為普遍表現在大腦組織中的調控次單位。PPP2R2B基因透過基因啟動子的差異使用及選擇性裁接，產生Bβ1和Bβ2兩種異構型蛋白。Bβ1啟動子上CAG三核苷酸重複擴增，可能因導致細胞質中Bβ1表現量的上升，而與第十二型脊髓小腦萎縮症相關。相反的，病例-對照組及啟動子的研究顯示，罕見短的CAG三核苷酸重複等位基因和低轉錄活性及阿茲海默氏症相關。為探討Bβ1在阿茲海默氏症上可能扮演的角色，本研究透過bisulfite處理及選殖定序，檢查5個阿茲海默氏症患者及年齡與性別配對的正常人，Bβ1啟動子1 kb片段的序列甲基化情形，結果發現患者的甲基化程度略高於正常人(雖然差異未達顯著性)，顯示外遺傳改變可能影響阿茲海默氏症患者Bβ1的表現。此外，本研究並用穩定誘導表現Myc標籤的Bβ1及Bβ2細胞株，來探討Bβ調節的PP2A在神經退化中的角色。結果發現表現Bβ2的細胞活性氧自由基增加。氧化劑TBH及Aβ1-40的添加更顯著提昇Bβ2表現細胞的活性氧自由基。Protein phosphatase 2A (PP2A) is the predominant serine/threonine phosphatase in eukaryotic cells. The PP2A holoenzyme is composed of scaffolding/structural A, regulatory/targeting B and catalytic C subunits. Bβ, a regulatory subunit encoded by the PPP2R2B gene, is widely expressed in neurons throughout the brain. Two major isoforms, Bβ1 and Bβ2, are produced through differential promoter usage and alternative splicing of the PPP2R2B gene. Increased expression of Bβ1 isoform due to CAG repeat expansion at the 5' end of the gene causes autosomal dominant spinocerebellar ataxia type 12. Contrarily, the case-control study and reporter assay revealed that the rare short low transcriptional activity alleles are associated with Alzheimer’s disease (AD). To study the roles of Bβ1 in AD, bisulfite sequencing was performed to assess the CpG 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 5' region of Bβ1 gene suggest that the epigenetic change may alter the Bβ1 expression in AD patients. In addition, Tet-on inducible cell lines expressing Myc-tagged Bβ1 and Bβ2 were used to study the role of Bβ-modulating PP2A in neuronal degeneration. The Bβ2-expressing cells are characterized by increase of reactive oxygen species (ROS). Addition of TBH (tert-butyl hydroperoxide) and Aβ1-40 also revealed increased ROS production in Bβ2-expressing cells.外遺傳甲基化阿茲海默氏症細胞模式氧化壓力PPP2R2BepigeneticDNA methylationAlzheimer’s diseaseoxidative stress