以大鼠神經細胞培養模式探討乙型類澱粉體對胰島素誘發Arc蛋白表現的影響及其機轉

The effects of amyloid-b on insulin-induced Arc expression and its underlying mechanisms in rat cortical neurons

何宣芳(Hsuan-Fang Ho)

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(2010年)

摘要

乙型類澱粉體(amyloid-beta，Aβ)是由位在細胞膜上的前驅蛋白amyloid precursor protein (APP)經酵素分解後的產物，具有維持神經細胞存活和促進神經先驅細胞分化的功能。但是當腦內Aβ含量增加時，就會開始不正常聚集而造成神經毒性，最終在細胞外堆積形成老年斑塊(senile plaque)，是阿茲海默氏症(Alzheimer’s disease，AD)病人腦中的典型神經病理學變化之一。AD是一種進行性神經退化性疾病，隨著病程發展，患者的認知功能包括學習與記憶能力逐步變差，終至喪失生活自理能力。諸多針對AD的研究即指出，Aβ的神經毒性會損害與學習記憶能力密切相關的突觸可塑性(synaptic plasticity)。文獻顯示，在執行學習與記憶以及維持長期突觸可塑性的過程中，有數種蛋白質會被大量合成，其中，Arc蛋白質(activity-regulated cytoskeleton associated protein)是早期基因(immediate early gene)的產物，會表現在具有活性的神經突觸上。Arc蛋白質的表現受到多種因素的調控，包括神經細胞的活動、神經滋養素(neurotrophins)的刺激、以及胰島素的作用等。過去的研究發現，當中樞神經系統裡的胰島素作用在海馬迴(hippocampus)及大腦皮質(Cortex)上時，可透過其訊息傳遞路徑改善認知功能。而當大腦中的胰島素及其訊息傳遞路徑受到損害時，則會造成與AD相似的神經退化性疾病。流行病學研究即指出第二型糖尿病是晚發型AD的危險因子之一。此外，因為AD同時表現出較低的中樞胰島素含量以及胰島素耐受性，被部分研究者視為是第三型糖尿病。雖然許多的證據都支持阿茲海默氏症與中樞神經系統中參與記憶形成的胰島素有關連，但其確實機轉並不完全清楚。因此本研究將探討Aβ是否會影響胰島素誘發Arc蛋白質表現的作用，並探討其分子機制。使用新生大鼠大腦皮質細胞初代培養進行實驗。結果發現，在胰島素作用下可誘發細胞產生Arc蛋白，呈時間及劑量相關，據此選擇胰島素400 nM處理2小時進行後續實驗。前處理MAPK傳遞路徑抑制劑(PD98059)或PI3K傳遞路徑抑制劑(Wortmannin)後，胰島素誘發Arc蛋白表現的現象受到明顯抑制，顯示胰島素可透過活化MAPK及PI3K傳遞路徑誘發Arc蛋白表現。而前處理非致死劑量A?珓寣A對胰島素誘發Arc蛋白表現亦有抑制效果，同時抑制p-Akt的表現量，但對於p-Erk表現無顯著影響，顯示A?珝|藉由干擾PI3K路徑的訊息傳遞過程而使Arc蛋白表現減少。本研究證實，過多的Aβ堆積會導致胰島素/接受器訊息傳遞路徑受損而使突觸可塑性功能喪失。這個結果很可能也是造成AD患者記憶功能退化的機轉之一。

關鍵字

胰島素 ； 乙型類澱粉體 ； 阿茲海默氏症

並列摘要

Amyloid-?? (A??) is a cleavage product of a large transmembrane protein, amyloid precursor protein. A?? has important physiological functions and may be crucial for neuronal cell survival. However, over-expression of extracellular A?? leads to the accumulation of A?? and the subsequent formation of senile plaques within the brain, which is a hallmark of neuropathological change in Alzheimer’s disease (AD) that is a progressive neurodegenerative disease characterized by a gradual decline in cognitive function. A?? aggregates or senile plaques can inhibit certain forms of synaptic plasticity that underlies learning and memory. Among various proteins related to synaptic plasticity, activity-regulated cytoskeleton-associated (Arc) protein is an immediate early gene product and plays a fundamental role in modulating synaptic plasticity. The expression of Arc protein is modulated by various stimulations including neuronal activities, neurotrophins actions, and insulin treatments. In the cerebral cortex and hippocampus, the activation of the insulin signaling pathway has been reported to improve cognition functions including memory formation. In contrast, impairments of the brain insulin and its signaling pathway result in the neurodegenerative changes similar to AD. Epidemiological studies reveal that type II diabetes mellitus is a risk factor for the development of late-onset AD. In addition, both reduced brain insulin and insulin resistance are found in AD patients, which evoke the suggestion that AD is type III diabetes mellitus. Although an association between the dysfunction of brain insulin and the occurrence of AD has been reported, the underlying mechanism is still uncertain. Therefore, in the present study, neonatal rat cortical neurons were used to investigate the effects of A?? on insulin-induced Arc expression, and the involved insulin signaling pathways would be confirmed to reveal the underlying mechanism. After treating cultures with insulin, the expression of Arc protein was increased in both time- and dose-dependent manners. The treatment of insulin at 400 nM for 2 h induced significant Arc expression and thus was selected for the following experiments. Pretreating cultures with PD98059 or Wortmannin significantly reduced insulin-induced Arc expression, indicating that insulin can induce Arc expression through activating the MAPK and PI3K signaling pathways. In addition, pretreating cultures with A?? at nonlethal level also significantly reduced insulin-induced Arc expression as well as the levels of p-Akt but not p-Erk. These results indicate that A?? can interrupt the activation of the PI3K/Akt signaling pathway leading to the reduction of insulin-induced Arc expression in rat cortical neurons. Therefore, this study demonstrates that over-expression of Aβ can impair the insulin signaling pathway and, thus, result in a reduction in insulin-induced Arc expression. This finding is possibly one of the mechanisms that induce memory loss in AD patients.

並列關鍵字

insulin ； amyloid-beta ； AD ； Arc

參考文獻

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