PPP2R2B過度表現對人類神經細胞骨架蛋白的影響

Abstract

蛋白質去磷酸酶2A型(PP2A)是一種由三種單體組成的酵素，它包含了一個催化單元(C)、一個結構單元(A)、以及一個可變的調節性單元(B)。這個可變的B單元(B, B',與B''家族)支配著PP2A的受質特異性及其在細胞內的表現位置，PP2A在細胞許多方面的功能都扮演了重要的角色。ppp2r2b是負責轉錄Bbeta可變單元的基因，Bbeta1與Bbeta2則是選擇性拼接下所轉譯出的兩種產物，它們具有不同的N端。第十二型脊髓小腦萎縮症 (SCA12)與ppp2r2b的5'-UTR產生過度擴增CAG三核苷酸有關。本研究目的是將胞器專一性的細胞質Bbeta1與粒線體Bbeta2等次單元，分別轉殖入神經纖維母細胞SK-N-SH，次將細胞經篩選之後，建立起永久表現的細胞株，維持其細胞生長，並使用西方墨漬法、共軛焦顯微鏡、PP2A酵素活性分析等方式深入探討這些轉殖細胞。轉殖細胞株皆分別過度表現Bbeta1與Bbeta2，其中Bbeta2的PP2A活性有顯著提昇，此外轉殖的Bbeta2共存於粒線體，Bbeta1則存在細胞質。Bbeta1與Bbeta2過度表現會造成tau過度表現與磷酸化，此現象與PI3-kinase/Akt/GSK-3beta pathway的活化相關，Bbeta1與Bbeta2過度表現也會破壞F-actin的完整性，影響adherens junction的分佈，而且 autophagosome生成也會增加。此外Bbeta2過度表現會促進神經分化，並且提昇Bcl-2表現。本研究證實所建立的神經退化性細胞模式，可做神經退化性疾病的發生及治療的參考。Protein phosphatase 2A (PP2A) is a heterotrimeric enzyme consisting of a catalytic subunit (C), a structural subunit (A), and a variable regulatory subunit (B). Variable B subunits (B, B', and B'' families) dictate PP2A substrate specificity and subcellular localization. PP2A plays an essential role in many aspects of cellular functions. ppp2r2b encodes Bbeta regulatory subunit, and Bbeta1 and Bbeta2 are two novel splice variant of the neuronal Bbeta regulatory subunit with divergent N-terminal tails. The CAG trinucleotide expansion in ppp2r2b 5'-UTR is involved in Spinocerebellar ataxias 12(SCA 12).We transferred neuron-specific Bbeta1 and Bbeta2 subunits into neuroblastoma cells SK-N-SH. The selected cells were established as permanent cell lines and maintained in media. Western blotting, confocal microscope, and PP2A activity assay are used to investigate the stably transfected cells. The PP2A activities were elevated in Bbeta2 cones. More experiments demonstrated that Bbeta1 and are Bbeta2 is colocalized in cytopasma and mitochondria, respectively. In addition, Bbeta1 and Bbeta2 overexpression in cones caused tau overexpression and phosphorylation that are associated with PI3-kinase/Akt pathway activation. Moreover, Bbeta1 and Bbeta2 overexpression abolishes the structure of F-actin, affects adherens junction distribution and increases the expression of Bcl-2 and autophagosome formation. Bbeta2 accelerates neuron differentiation. The work provided a cell model for studying the onset of neuron disease pathogenesis and possible therapy.