PPP2R2B基因：啟動子記述及Bβ1/Bβ2 isoform在神經退化的角色

PPP2R2B gene: promoter characterization and role of Bβ1/Bβ2 isoform in neurodegeneration

林志信(Chih-Hsin Lin)

指導教授 ： 李桂楨

國立臺灣師範大學/理學院/生命科學研究所/博士(2010年)

摘要

PPP2R2B (亦稱為Bβ)廣泛表現在神經元，可調節去磷酸酶PP2A對tau及其他受質的去磷酸化作用。PPP2R2B基因5'端CAG重複擴增，導致體染色體顯性遺傳的神經退化性疾病第十二型小腦萎縮症。相反地，低轉錄活性且稀有短的等位基因，與台灣阿茲海默氏病和原發性顫抖症相關。本論文的第一部分在探討PPP2R2B基因CAG重複序列的角色，和鄰近cis要素及相關蛋白質調控PPP2R2B表現的情形。缺失/特定點突變、電腦模擬、cDNA 過度表現等實驗顯示，CREB1和SP1結合在CAG 重複的上游保守序列，來增加PPP2R2B的表現；然而TFAP4結合在CAG 重複的下游保守序列，來降低PPP2R2B的表現。DNA pull-down和染色質免疫沉澱-聚合酶連鎖反應等實驗進一步證實，CREB1、SP1和TFAP4結合在PPP2R2B起動子上。AT重複置換CAG重複的實驗顯示，CAG重複本身亦為一正調控PPP2R2B表現的cis要素。這些結果顯示CREB1、SP1和TFAP4在調節PPP2R2B表現中扮演角色，因此提供了一個調節PP2A活性的機制。論文的第二部分著重在建立穩定誘導表現Myc標籤的Bβ1/Bβ2細胞株，來探討Bβ調節的PP2A在神經退化中所扮演的角色。被誘導的Bβ1和Bβ2蛋白分別座落在細胞質和粒線體。Bβ細胞株的細胞週期分析顯示Bβ2表現的細胞，subG1和G2/M顯著增加，並伴隨著細胞存活率的顯著降低及cell division cycle (Cdc2)磷酸化的增加。表現Bβ2的細胞其特徵包括：活性氧自由基(ROS)和caspase 3活性增加、粒線體膜電位降低、Bax增加和cytochrome c由粒線體釋出至細胞質等，暗示Bβ2會誘導細胞自戕。抗氧化劑-tocopherol的添加可減弱ROS產生及cytochrome c釋放。總言之，這些結果顯示Bβ2能透過提升粒線體ROS的產生而誘導細胞凋亡。因此抑制Bβ2表現可能被發展為有潛能的治療策略，來治療與PP2A/Bβ2活性增加相關的神經退化性疾病。

關鍵字

小腦萎縮症第十二型 ； PPP2R2B ； 啟動子 ； CREB1 ； SP1 ； TFAP4 ； 細胞週期 ； 活性氧 ； 细胞凋亡 ； 抗氧化劑apoptosis, a

並列摘要

PPP2R2B (also known as Bβ), a protein widely expressed in neurons, regulates the protein phosphatase 2A (PP2A) activity for dephosphorylation of tau and other substrates. CAG repeat expansion at the 5' end of the PPP2R2B gene causes autosomal dominant spinocerebellar ataxia type 12. Contrarily, the rare short alleles with low transcriptional activity are associated with Taiwanese Alzheimer's disease and essential tremor. In the first part of the dissertation, the roles of CAG repeats and the flanking cis elements and the associated proteins in controlling PPP2R2B expression were investigated. Deletion/site-directed mutagenesis, in silico searches and cDNA overexpression revealed that CREB1 and SP1 bind to the conserved sequence upstream the CAG repeats to up-regulate PPP2R2B expression, whereas TFAP4 binds to the conserved sequence downstream the CAG repeats to down-regulate PPP2R2B expression. The binding of CREB1, SP1 and TFAP4 to the PPP2R2B promoter was further confirmed by DNA pull-down and ChIP-PCR assays. CAG repeats itself also functions as a cis element to up-regulate PPP2R2B expression as AT repeat length has no effect on PPP2R2B expression. This data provide evidence that CREB1, SP1 and TFAP4 play roles in modulating PPP2R2B expression, thus offering a mechanism of regulating PP2A activity. The second part of the thesis is to uncover the role of Bβ-modulating PP2A in neuronal degeneration using stably induced cell models expressing Myc-tagged Bβ1 and Bβ2. The induced Bβ1 and Bβ2 proteins were mainly localized in cytoplasm and mitochondria, respectively. Cell cycle analysis of Bβ cell lines revealed a significant increase in the cell population at subG1 and G2/M phases in Bβ2-expressing cells, along with significant reduced cell viability and increased inhibitory phosphorylation of cell division cycle 2 (Cdc2). The Bβ2-expressing cells are characterized by increase of reactive oxygen species (ROS) and caspase 3 activity, loss of mitochondrial membrane potential, as well as Bax increase and cytochrome c release, all of which implicate elevated apoptosis. Addition of an antioxidant -tocopherol attenuated ROS production and cytochrome c release. Taken together, these results suggest that Bβ2 is able to induce apoptosis via enhancing mitochondrial ROS production. Thus inhibition of Bβ2 expression may be an attractive avenue for developing potential therapeutic strategies to treat neurodegenerative disorders related to increased PP2A/Bβ2 activity.

並列關鍵字

spinocerebellar ataxia type 12 ； PPP2R2B ； promoter ； CREB1 ； SP1 ； TFAP4 ； cell cycle ； reactive oxygen species ； apoptosis ； antioxidant

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