環境壓力對於神經母細胞過度表現細胞質或粒線體PPP2R2B之影響

Abstract

Protein phosphatase 2A (PP2A)是serine/threonine去磷酸酶的一種，能調節細胞的生長、存活和凋亡。哺乳類動物中，Bβ次單位PPP2R2B是PP2A在腦神經細胞中的專一表現的次表型。當 Bβ基因有所缺失或過度表現時，會造成神經細胞的病變。本研究使用可以持續且穩定過度表現細胞質PPP2R2B蛋白(Bβ1)及粒線體 PPP2R2B蛋白(Bβ2)的神經細胞株，觀察飢餓壓力、低氧環境和Tunicamycin(TM)等藥物引起的內質網壓力，對細胞表型的影響。 研究結果顯示Bβ1細胞在飢餓壓力下，其粒線體膜電位及存活率都會下降，而sub-G1期比例也有顯著提升，代表產生細胞凋亡。但若以細胞自噬抑制劑Bafilomycin A1預處理，則會抑制因飢餓壓力引起的細胞自噬，進而減緩後續的細胞凋亡，但3-methyladenine (3-MA)則無此效果。 另一方面，Bβ2細胞株的存活率會因為受到內質網壓力而有下降，由流式細胞儀及螢光免疫染色發現細胞內的酸性液泡會增多，代表有細胞自噬增加，但Bβ1細胞株則否。但以細胞自噬抑制劑3-MA先對細胞作前處理24小時後，再施以內質網壓力，則各Bβ2細胞株的sub-G1 期比例皆會降低，而酸性液泡胞器的密度也明顯降低。 此外，在低氧壓力對於過度表現PPP2R2B的蛋白，無論是細胞質或粒線體，皆會造成sub-G1 期上升，引發不同程度的細胞凋亡。 本論文針對過度表現PPP2R2B的細胞株分別由飢餓壓力、內質網壓力和低氧環境下，觀察細胞所產生的表型變化，模擬PPP2R2B過度表現之神經退化病變的產生，並瞭解病變機制，祈能對PPP2R2B相關疾病的研究與治療有所助益。

Protein phosphatase 2A (PP2A) is one of four major classes of serine/threonine phosphatases. The alternatively splicing of brain-targeted regulatory subunit PPP2R2B encodes two regulatory subunit isoforms, cytoplasmic-specific Bβ1 and mitochondria-specific Bβ2. The two constructs were transfected into human neuroblastoma cells, SK-N-SH, respectively, and the stable clones over-expressing either Bβ1 or Bβ2 established. To address the onset of neuropathogenesis under different environmental stress, Bβ1 and Bβ2 clones were exposed to starvation (HBSS), and endoplasmic reticulum (ER) stress and hypoxia, respectively. To test how Bβ clones respond to starvation, the cells were exposed to Hank's Buffered Salt Solution (HBSS). Cell death was induced Bβ1 clones, but not Bβ2 clones, following 6 hr starvation. Suppression of autophagy using pharmacological inhibitor, Bafilomycin A1, can rescue autophagic cell death and reduce autophagy-related apoptosis. In contrast, 3-MA (3-methyladenine) did not rescue autophagy-related apoptosis. To test the cells under ER stress, the cells were treated with 0.1 μg/ml of N-glycosylation inhibitor, tunicamycin (TM), in which cell death was observed in Bβ2 clones following 48 h treatment, but not in Bβ1 clones. The analysis of distribution and intensity of LysoTracker and GFP-LC3 staining by fluorescence microscopy showed that TM induced autophagolysosome formation in Bβ2 cells prior to apoptosis. The autophagic cell death in Bβ2 clones cell can be rescued by autophagy inhibitor, 3-methyladenine (3-MA). On the other hand, cells incubated in a hypoxia chamber (0.5% O2, 5% CO2) for more than 48hr showed increased sub- G1 distribution not only in Bβ1 clones, but also in Bβ2 clones. More sub-G1 phase cells appeared in Bβ2 clones than in Bβ1 clones increased with the in time under hypoxia. Therefore, we showed that cells with ectopically expressed regulatory subunit PPP2R2B of holoenzyme PP2A were predisposed to differential autophagy related cell death. Starvation induced autophagic cell death in Bβ1 clones with overexpressed cytoplasmic PPP2R2B, where ER stress induced autophagic cell death in Bβ2 clones with mitochondrial PPP2R2B. The result promised a model for studying the mechanism and function of aberrant PPP2R2B expression in neuronal cells under starvation.