PC12細胞和大鼠胚胎神經細胞內鋅離子濃度上升在多巴胺活化細胞自噬所扮演的角色

The Roles of Intracellular Zinc Elevation in the Dopamine-induced Autophagy Activation in PC12 Cells and Cultured Rat Embryonic Cortical Neurons

洪慧馨(Hui-Hsing Hung)

指導教授 ： 潘建源

國立臺灣大學/生命科學院/生命科學系/博士(2016年)

https://doi.org/10.6342/NTU201610062

摘要

在大腦中，含有豐富的微量金屬鋅離子，雖然鋅離子為維持神經活性所必需，但如果濃度太高，也會引起毒性，對神經細胞造成傷害。多巴胺是一種重要的神經傳導物質，同時也參與細胞自噬、細胞凋亡和神經調節。細胞自噬雖會導致神經細胞死亡，但也能增加神經細胞的存活率。目前對於多巴胺在神經退化性疾病中，是否造成神經細胞死亡仍不甚了解。在此研究中，我們先研究大鼠腎上腺髓質嗜鉻細胞瘤細胞和大鼠胚胎神經細胞在處理高濃度的多巴胺和鋅離子情況下，細胞自噬所扮演的角色。之後，我們研究多巴胺如何引起細胞內鋅離子濃度上升，進而造成細胞自噬。當細胞處理高濃度的多巴胺和鋅離子，會造成EGFP-LC3點狀螢光聚集數目有顯著性的增加，並且也增加內生性LC3脂化程度，也就是細胞自噬的程度增加。對大鼠腎上腺髓質嗜鉻細胞瘤細胞處理phosphatidylinositol 3-kinase的抑制劑，或者使用siRNA減少細胞內Atg7蛋白質的量，點狀螢光聚集數目的增加會被顯著性地抑制。除此之外，抑制細胞自噬小體的形成，會讓更多大鼠腎上腺髓質嗜鉻細胞瘤細胞進行細胞凋亡。不論是多巴胺或是鋅離子的處理都會顯著性增加細胞內鋅離子的濃度，然而，如果在處理多巴胺或鋅離子之前先讓細胞在有鋅離子螯合劑的環境中，多巴胺處理造成的細胞自噬增加則會被顯著性的抑制。在神經細胞中，利用多巴胺第一型受器、cAMP-dependent protein kinase (PKA) 和一氧化氮合成蛋白的抑制劑，可以抑制多巴胺造成的鋅離子濃度增加。反之，利用PKA活化劑和直接給予一氧化氮則可以增加細胞內的鋅離子濃度。收集細胞的所有蛋白質，並將其依照分子量分類，只有分子量介於五千道爾頓和一萬道爾頓間的蛋白質，能在一氧化氮的處理下，釋放鋅離子。除此之外，處理鋅離子的螯合劑也能抑制一氧化氮所引起的細胞自噬。因此，我們的結果顯示在高濃度的多巴胺和鋅離子處理下，會活化細胞自噬，而細胞自噬在此扮演保護的角色。我們也證明了多巴胺是藉由活化多巴胺第一型受器，造成PKA的活化，產生一氧化氮，引起細胞內鋅離子濃度上升，而此鋅離子濃度上升為引起細胞自噬所必需。

關鍵字

鋅離子 ； 多巴胺 ； 細胞自噬 ； 一氧化氮 ； 神經細胞

並列摘要

Zinc ion (Zn2+), one of the most abundant trace metals in the brain, is essential for neuronal activities but induces toxicity when the concentration is abnormally high. Dopamine is an important neurotransmitter and is involved in autophagy, apoptosis, and neuromodulation. Autophagy plays an important dual role in neuronal cell death and cell survival. However, the mechanism behind the toxic effects of dopamine in neurodegenerative diseases is not clear. In this report, we first characterized how the high concentrations of dopamine and Zn2+ induced autophagy in PC12 cells and cultured embryonic cortical neurons; then investigated how dopamine elevated the intracellular Zn2+ concentration ([Zn2+]i) for autophagy activation. High concentrations of dopamine and Zn2+ increased the number and size of the aggregates of EGFP-LC3 expressed in cells as an indicator of autophagosome formation. The Western blot analysis showed the lipidation level of LC3 increased by these treatments in PC12 cells and cultured neurons. Introducing siRNA against ATG7, an initiator protein of autophagy, and blocking the phosphatidylinositol 3-phosphate kinase inhibited the formation of EGFP-LC3 aggregates in PC12 cells. In addition, blocking autophagosome formation increased the level of phosphatidylserine exposure on the outer membrane leaflet in PC12 cells when treated with dopamine or Zn2+. Dopamine or Zn2+ treatment significantly elevated [Zn2+]i; however, pretreatment of PC12 cells and neurons with a Zn2+ chelator suppressed the dopamine-induced autophagosome formation and LC3 lipidation. In neurons, inhibitors against the dopamine D1-like receptor, cAMP-dependent protein kinase (PKA), and NO synthase suppressed the dopamine-induced [Zn2+]i elevation. PKA activators and NO generators directly elevated the [Zn2+]i in cultured neurons. Using cell fractionation, proteins with M.W. values between 5 and 10 kDa were found to release Zn2+ following NO stimulation. In addition, chelating the [Zn2+]i elevation blocked the NO-activated autophagy. Therefore, our results indicate that treating cells with dopamine and Zn2+ results in the activation of the autophagy pathway in an effort to enhance cell survival. Furthermore, we demonstrate that the dopamine-induced [Zn2+]i elevation is mediated by the D1-like receptor-PKA-NO pathway and is crucial for autophagy activation.

並列關鍵字

Zn2+ ； dopamine ； autophagy ； NO ； neuron

參考文獻

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