利用有對熱逆境敏銳反應的腦細胞來探討轉譯抑制的機制

Abstract

細胞受到刺激造成不能正常摺疊的蛋白質累積在內質網 (Endoplasmic Reticulum, ER) 而產生內質網壓力 (ER stress) 時，C/EBP homologous protein (CHOP) 會受unfolded protein response (UPR) 調控，並在細胞修復與凋亡(apoptosis)過程中扮演著重要的角色。目前已知 ER stress 會調節 chop mRNA 的 upstream open reading frame (uORFchop) 的轉譯抑制能力來影響 CHOP 表現，但其詳細調控模式仍不清楚。因此本研究將利用 CMV promoter 驅動表現末端融合綠螢光報導基因的人類 uORFchop (huORFchop) 之轉殖斑馬魚 (huORFZ) 做為研究平台，在 in vivo系統下研究uORFchop 的調控機制。以熱逆境誘導 ER stress 產生時，96 hpf 時期之 huORFZ 胚胎的腦部綠螢光訊號強度與熱處理時間和胚胎損傷程度有正相關的趨勢，說明 huORFZ 胚胎在一群對於熱有敏銳反應的腦細胞，且部分位於腦腔 (ventricular zone) 與腦摺 (brain folds) 周圍，而活化態 caspase 3的免疫螢光染色結果顯示這些綠螢光腦細胞不會走向凋亡。接著在各種標記蛋白的免疫螢光染色結中，因熱逆境誘導產生的綠螢光訊號會出現在 GS(+) 似放射狀神經膠細胞 (redial glia-like cells)，而不出現於 HuC/D(+) 神經細胞 (neurons)，表示這兩種腦細胞對 ER stress 的反應路徑有些不同，因此熱逆境只會誘導GS(+) 神經膠細胞的 huORFchop 失去功能。為探討綠螢光細胞所屬特性，利用 BrdU 標定法指出少數綠螢光細胞在熱處理後的24小時內有增生現象，另一方面則藉著熱逆境產生的綠螢光追蹤到極少部分綠螢光細胞在9天大的胚胎發育時期會表現 HuC/D，表示這些對熱敏感的綠螢光腦細胞可能具有增生與分化潛力。更進一步地，利用結合活體胚胎冷凍包埋切片與 Laser Microdissection 所建立的細胞收集系統，分別擷取有經過熱處理的 huORFZ 胚胎之非綠螢光腦細胞與綠螢光腦細胞後，藉由 microarray 分析來尋找影響 huORFchop 功能的相關因子。Microarray 結果指出基因 dkey 在綠螢光細胞內的轉錄效率較非綠螢光細胞高，而全胚胎原位雜交染色發現 dkey mRNA 表現量與具有熱敏銳特性之神經膠細胞的綠螢光訊號有正相關的趨勢。此外，在沒有將胚胎進行熱處理的情況下，有注射 dkey mRNA 的 huORFZ 胚胎會出現綠螢光訊號。綜合上述實驗結果，本研究證明了熱逆境誘導 huORFchop轉譯抑制功能的喪失具有細胞種類特異性，以及推測 dkey 可能參與在熱逆境影響 huORFchop 的調控路徑中。

When cells encounter endoplasmic reticulum (ER) stress caused by the accumulation of misfolded proteins in the ER, unfolded protein response (UPR) lead to upregulation of C/EBP homologous protein (CHOP) which plays important roles in both cell survival and apoptosis. It has been reported the uORF sequence located in 5`UTR of human chop gene (huORFchop) inhibits the translation of chop. However, underlying molecular mechanisms is poorly understood and there was no in vivo animal model for studying uORFchop mechanism until recently. To study the mechanism of huORFchop mediated translational control in vivo, I used the zebrafish transgenic line, termed huORFZ, harboring a construct in which the uORFchop sequence is added to the leader of GFP and is driven by a cytomegalovirus promoter. The GFP appeared only when huORFZ embryos were treated with ER stress. Through using the heat-shock to induce ER stress, the expression level of GFP signal in the 96 hpf huORFZ embryo brain was dependent to treatment time, indicating dose-dependent effect. Also, GFP signal appeared in ventricular zone and brain folds. However, Immunohistochemistry of active caspase3 showed that these GFP(+) brain cells as non-apoptotic cells. Interestingly, immunohistochemistry identified these heat-induced GFP(+) cells with longer processes were GS(+) redial glia-like cells, but not HuC/D(+) neurons, suggesting the responses to ER stress were different between these two groups of cells and huORFchop mediated translational inhibition was only repressed by heat-shock in GS(+) glia cells. Moreover, BrdU assay showed that GFP(+) cells proliferated in 24 hr after heat-shock treatment. Lineage tracing by heat-induced GFP showed a few GFP(+) cells expressed neuron-specific marker HuC/D in 9 dpf embryo. Therefore, the heat-induced GFP(+) brain cells may have proliferational and deffriancational potential. Furthermore, I developed a Laser capture Microdissection to harvest GFP(-) brain cell and GFP(+) brain cells, and using microarray analysis to find out which factors are involve in regulating huORFchop. In the microarray results, dkey was one of the upregulated genes in GFP(+) cells. Whole mount in situ hybridization showed that the translation of dkey was up regulated and its translation level correlated to the GFP signal in embryo brain. Moreover, GFP signal were detected in huORFZ embryos injected with dkey mRNA without heat-shock treatment. Based on the above results, I demonstrated that repression of huORFchop mediated translational inhibition have cell-type-specific response to heat stress and propose that dkey is involved in huORFchop mediated translational control under heat-shock stress.