鋁暴露降低腦泌神經滋養素誘發Arc蛋白表現的可能機轉

Possible mechanisms for the reduction of BDNF-induced Arc expression caused by aluminum maltolate

鄭琇敏(Hsiu-Min Cheng)

指導教授 ： 陳贊如

高雄醫學大學/醫學院/生理及分子醫學研究所/碩士(2009年)

摘要

目前已經證實鋁暴露和數種神經退化性疾病包括阿茲海默氏症有密切關連。雖然已有多篇研究證實鋁暴露會造成學習與記憶能力的損害，但確切的機轉仍然未知。先前的研究已證實，腦泌神經滋養素(BDNF)可藉由調節神經元/突觸可塑性而參與記憶形成的過程。在BDNF的多種下游因子中，早期基因Arc的蛋白質產物是協助突觸鞏固的重要因子，因此BDNF誘發Arc 蛋白表現可作為研究神經元/突觸可塑性的模式。在本研究中，將針對鋁暴露干擾BDNF誘發Arc蛋白表現的可能機制進行探討。分化後的人類神經母細胞瘤細胞，SH-SY5Y細胞，給予非致死劑量的aluminum maltolate (Al(malt)3, 200µM)前處理24小時，再給予BDNF (10 ng/ml)一小時誘發Arc蛋白表現；由西方點墨法的結果發現，在Al(malt)3前處理後，BDNF誘發的Arc蛋白產量明顯減少，此外，BDNF誘發的磷酸化Akt含量沒有變化，但磷酸化Erk的含量則會減少，顯示在非致死劑量的Al(malt)3作用下，BDNF活化PI3K/Akt路徑的程度不受影響，但活化MAPK/Erk路徑的程度則會降低。由於突觸部位的轉譯作用和神經元/突觸可塑性密切相關，因此同時檢視涉及轉譯起始步驟的相關因子活化程度的變化，結果發現，PI3K/Akt的下游因子mTOR的磷酸化在BDNF作用下會增加但不受Al(malt)3前處理的影響。mTOR的下游因子4EBP的磷酸化在BDNF作用下也會增加但Al(Malt)3前處理會使其增加幅度減少，eIF4E的磷酸化在BDNF作用下也是增加而且在Al(malt)3前處理的影響下會減少但並沒有明顯降低，顯示主要變化發生在4EBP。雖然mTOR磷酸化不受Al(malt)3前處理的影響，但由於Erk也會影響4EBP的磷酸化，因此4EBP的變化與Al(malt)3降低BDNF誘發Erk磷酸化的結果是相符的。在Al(malt)3前處理下，BDNF誘發的4EBP磷酸化會降低，使得4EBP較難與eIF4E脫離，因此和4EBP結合在一起的eIF4E無法去啟動轉譯的起始步驟，這極可能就是Al(malt)3降低BDNF誘發Arc蛋白表現的細胞機轉之一。

關鍵字

鋁暴露 ； 神經元/突觸可塑性 ； 腦泌神經滋養素 ； Arc蛋白

並列摘要

Several neurodegenerative diseases including Alzheimer’s disease have been suggested to be closely related to aluminum exposure. Although previous studies have confirmed the impairment of learning and memory following aluminum exposure, its underlying mechanism is still uncertain. Brain-derived neurotrophic factor (BDNF) is well known to be involved in memory formation by regulating neuronal/synaptic plasticity. Recently, an immediate early gene Arc (activity-regulated cytoskeleton-associated gene) is found to be a downstream effector of BDNF and its protein production is involved in neuronal/synaptic plasticity. Thus, BDNF-induced Arc expression has been regarded as a useful model to investigate the cellular mechanism of neuronal/synaptic plasticity. In this study, an effort was made to evaluate the effect of aluminum on BDNF-induced Arc expression and its possible underlying mechanisms. Differentiated human neuroblastoma SH-SY5Y cells were treated with aluminum maltolate (Al(malt)3) at non-lethal dose (200 ?嵱) for 24 h followed by BDNF (10 ng/ml) for 1 h to induce Arc expression. Western blot analysis showed that Al(malt)3 pretreatment significantly reduced BDNF-induced Arc expression. Simultaneously, Al(malt)3 pretreatment significantly reduced BDNF-induced phosphorylation of Erk but not Akt, indicating that non-lethal Al(malt)3 only interfered with the activation of MAPK/Erk but not PI3K/Akt signaling pathway following BDNF induction. Since the local translation at active synapses is closely related to neuronal/synaptic plasticity, the phosphorylation states of various factors involved in the translation initiation step were evaluated. The phosphorylation/activation of mTOR, a downstream effector of the PI3K/Akt pathway, was increased by BDNF treatment, which was not influenced by Al(malt)3 pretreatment. However, the phosphorylation/inactivation of 4EBP, a downstream effector of mTOR, was also increased by BDNF treatment, which was reduced by Al(malt)3 pretreatment. As for the eIF4E, an important initiation factor, its phosphorylation/activation was increased by BDNF treatment, which was reduced by Al(malt)3 pretreatment. These results indicate that the major effect of Al(malt)3 pretreatment was on the phosphorylation state of 4EBP. Even though Al(malt)3 pretreatment did not alter BDNF-induced phosphorylation of mTOR, it did reduce BDNF-induced Erk phosphorylation, which may account for the reduction of BDNF-induced 4EBP phosphorylation since previous evidence has shown that p-Erk can also promote the phosphorylation of 4EBP. Furthermore, when 4EBP binds to eIF4E, the release and phosphorylation/activation of eIF4E are prevented and lead to a failure of translational initiation. BDNF can phosphorylate/inactivate 4EBP and then facilitate the initiation step. Therefore, in the present study, non-lethal Al(malt)3 pretreatment may reduce BDNF-induced Arc expression through interfering with the activation of MAPK/Erk pathway and the subsequent phosphorylation of 4EBP leading to an impaired translational initiation step.

並列關鍵字

aluminum exposure ； neuronal/synaptic plasticity ； BDNF ； Arc protein

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