不同型態的神經源細胞對於不同ER-associated Stress有細胞專一性的逆境反應

Abstract

在in vitro狀態下，腦部組織細胞遭遇到環境壓力，像是熱誘導( heat shock )或是酒精(alcohol stress)等內質網相關壓力(Endoplasmic reticulum-associated stress)，會開啟內質網逆境反應(ER stress response)來應對逆境。然而目前研究對於腦部組織不同細胞種類，在heat shock 或alcohol stress處理後的in vivo反應的情況缺乏探討。因此本篇研究利用，huORFZ基因轉殖斑馬魚品系作為實驗模式動物，來觀察在in vivo情況下，受到heat shock 或 alcohol stress處理後不同種類的腦細胞的反應是否有差異。huORFZ基因轉殖斑馬魚，為利用CMV promoter驅動，帶有人類chop 基因5’ UTR 的upstream open reading frame (huORFchop)，後接上GFP報導基因的表現載體的斑馬魚品系。利用免疫螢光染色標定GFP實驗，證實在正常情況下，huORFZ胚胎的報導基因GFP蛋白質表現完全會受到huORFchop抑制；在對huORFZ胚胎處以heat shock 或alcohol stress後，觀察到在腦部組織皆會被誘導出GFP 表現。在利用whole mount in situ hybridization 也發現斑馬魚内生性ER stress response genes perk、 xbp-1、 atf3和 zfchop的 mRNAs 會在腦部組織增量表現，顯示腦部組織有壓力反應(stress response)。利用免疫螢光染色標定星狀細胞(Astrocytes)特有的GFAP marker或神經細胞(Neurons)特有的HuC marker，來分析腦部這些GFP-positive的細胞種類。發現heat shock所誘導的GFP-positive細胞部分為星狀細胞，而非神經細胞；但由alcohol stress所誘導的GFP-positive細胞部分則為星狀細胞及神經細胞。利用fluorescence in situ hybridization (FISH)實驗，標定星狀細胞特有gfap mRNA marker，或神經細胞特有huc mRNA marker，然後分析在腦部組織增量表現的那些內生性zfchop mRNA細胞種類：發現被誘導增量表現的zfchop mRNA細胞種類，在heat shock處理後部分也是星狀細胞，而非神經細胞；而在alcohol stress 處理後部分也是星狀細胞及神經細胞。顯示，在星狀細胞和神經細胞對於熱誘導及酒精不同的逆境，是具有細胞特異性。接著利用免疫螢光染色標定受逆境誘導表現的GFP-positive細胞及TUNEL assay 標定 apoptosis細胞：發現heat shock及alcohol stress所誘導的GFP-positive細胞，絕大多數不為apoptotic細胞；即使以更強的heat shock逆境處理huORFZ胚胎，其所誘導的GFP-positive細胞，(>90%)均不為apoptotic細胞。顯示絕大多數GFP-positive細胞不會發生apoptosis，並不是因為逆境強度不夠而造成。另一方面，在70 min的heat shock 逆境處理huORFZ胚胎中，可觀察到原先在60 min的逆境強度會被誘導表現綠螢光的細胞外，有更多的細胞會隨著逆境強度的增強而被誘導表現綠螢光。顯示先表現綠螢光的細胞，比起因逆境強度增強而被誘導表現綠螢光的細胞；及會被誘導表現綠螢光的細胞比未凋亡但沒有表現綠螢光的細胞，對於逆境的反應是更為stress-sensitive。綜合以上結果發現，星狀細胞及神經細胞，對於heat shock 或alcohol stress具有細胞專一性的壓力反應，且在in vivo情況中，不同逆境處理之下會誘導不同種類細胞有壓力反應。而heat shock 或alcohol stress所誘導表現的綠螢光細胞，絕大部分不會發生apoptosis，且表現綠螢光的細胞是一群對逆境較為敏感，具有stress-sensitive特性的細胞。

It has been reported that the brain cells which are exposed in vitro to the environmental stresses such as heat-shock and alcohol induce the ER stress response. However, there is no in vivo evidence to demonstrate whether the different cell-types of brain tissue respond under heat-shock and alcohol stresses. To address this issue, we employed a transgenic line of zebrafish we generated, designated as huORFZ, in which the upstream open reading frame located at 5’UTR of human chop is added to the leader of GFP and driven by a cytomegarovirus promoter. Using immunostaining, we proved that the translation of GFP in embryos derived from huORFZ was completely inhibited during normal condition. But, when embryos were treated with either heat-shock or alcohol, the GFP reporter was induced in the brain. In addition, the transcripts of endogenous ER-stress-response genes such as perk, xbp-1, atf3, and zfchop were increased in the brain of huORFZ embryos treated with stresses. Furthermore, we found that the GFP-positive cells in hindbrain induced by heat-shock were astrocytes only; whereas the GFP-positive cells in hindbrain induced by alcohol were astrocytes and neurons. This result was consistent with performing fluorescence in situ hybridization using astrocytes-specific gfap marker and neurons-specific huc marker, indicating that astrocytes and neurons respond to heat-shock and alcohol stresses in a cell-type specific manner. We also confirmed that most of the GFP-positive cells (over 90%) induced by either heat-shock or alcohol were not apoptotic cells. These induced GFP-positive cells did not go further apoptosis even when embryos were treated with more severe heat- shock. Interestingly, we found that the number of GFP-positive cells increased when more severe heat-shock was employed, suggesting that the GFP-positive cells induced by low strength of stress might be more stress-sensitive than those alive GFP-negative cells. Taken together, we concluded that different cell-types in brain are respondent to different ER-associated stresses, and the induced non-apoptotic GFP-positive cells might be a group of cells having stress-sensitivity.