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標題: | 神經傳遞物質增加胞內鋅離子濃度與神經發炎反應在初代培養大鼠神經細胞的關聯 Investigation into Neurotransmitter-Induced Intracellular Zn2+ Elevation and its Correlation with Inflammatory Response in Primary-Cultured Rat Embryonic Cortical Neurons |
作者: | 曾惠群 Hui-Chiun Tseng |
指導教授: | 潘建源 Chien-Yuan Pan |
關鍵字: | 多巴胺,麩胺酸,發炎反應,神經退化性疾病,一氧化氮,一氧化氮合成酶,鋅離子, dopamine,glutamate,inflammatory response,neurodegeneration,nitric oxide,nitric oxide synthase,Zn2+, |
出版年 : | 2024 |
學位: | 博士 |
摘要: | 離子恆定參與調節許多重要生理功能,如鐵離子參與在氧氣的運輸,鈉鉀離子參與動作電位的變化,以及鈣離子調控神經傳遞物質的釋放和肌肉的收縮等等。而鋅離子常作為輔酶維持蛋白質與酵素的功能,同時也是許多蛋白據以維持結構的重要因子。近年研究發現有多種鋅離子轉運蛋白與結合蛋白,可嚴格調控細胞中鋅離子的恆定,而鋅離子恆定失調可導致細胞凋亡甚至神經退化等疾病。先前研究顯示,帕金森氏症患者腦中黑質的鋅離子濃度較健康者高,顯示高濃度的鋅離子可能對神經有害。然而帕金森氏症的研究,多集中於探討多巴胺 (dopamine, DA)氧化所產生活性氧類物質破壞粒線體,或是鋅離子影響粒線體功能,導致神經細胞死亡與神經退化;然而我們實驗室先前的研究成果顯示,DA會透過D1-like 受體活化NO合成酶 (NOS) 產生一氧化氮,提升胞內鋅離子濃度 ([Zn2+]i),以啟動細胞自嗜導致離體培養的大鼠胚胎皮質神經細胞死亡。而另一種常見的興奮性神經傳遞物質麩胺酸 (glutamate, Glu),已知也可活化NOS,但不知Glu是否亦可增加 [Zn2+]i。
近年研究發現,發炎反應是神經退化的原因之一。本研究中,我們首先使用D1-like 受體的促進劑 DHX,證實在初級培養的大鼠胚胎神經細胞中,並非DA的氧化,而是透過D1-like 受體造成 [Zn2+]i 上升,而發炎抑制劑可減少多巴胺鋅離子所造成的神經細胞死亡;且DA透過 [Zn2+]i 上升,增加NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3)發炎小體的形成;除增加NLRP3的表現量,也使下游Caspase-1活化,導致interleukin-1 beta (IL-1β)及Gasdermin D (GSDMD)成熟。且分解後的N端GSDMD,並沒有到細胞膜,而主要是到自嗜小體上,當我們以IL-1β預處理神經細胞,可降低DA造成的神經細胞死亡。這些成果顯示輕微的發炎反應與神經細胞中細胞自嗜有交互作用,影響細胞存活。 而以Glu刺激培養的神經細胞,我們發現會透過離子型Glu受體,經由鈣離子,活化鈣調素/鈣調素依賴蛋白激酶 II ,短期增加NOS磷酸化,以合成NO,導致 [Zn2+]i 上升。且短時間內並不會破壞粒線體的活性,但卻增加了NLRP3發炎小體的數量,而Zn2+ 螯合劑,TPEN的預處理可抑制發炎小體的產生。因此維持鋅離子恆定,是防止神經細胞誘發發炎反應導致死亡的關鍵因素。 不同的神經傳導物質,利用各自的途徑,活化NOS產生NO,使 [Zn2+]i上升,以啟動細胞自嗜、發炎反應等機制,進行交互作用而導致細胞死亡。這些結果顯示神經傳導過程中,鋅離子是一個重要的訊息分子,調控神經細胞長期活性變化;因此了解如何控制鋅離子恆定以調節自嗜及發炎,可為神經退化性疾病的治療,提供新穎的發展策略。 Ion homeostasis is involved in many critical physiological functions. For instance, iron participates in oxygen transport, sodium and potassium modulate the action potentials in neurons, and Ca2+ regulates neurotransmitter release and muscle contraction. Zinc ion (Zn2+) serve as co-factors to maintain the functions and structures of proteins and enzymes. Recent studies have discovered multiple Zn2+ transporters and binding proteins that strictly regulate the steady-state of the intracellular Zn2+ concentration ([Zn2+]i) within cells. Disruption in Zn2+ homeostasis has been linked to cell apoptosis and neurodegenerative diseases. Some studies have shown higher Zn2+ level in the substantia nigra of Parkinson''s disease (PD) patients compared to healthy individuals, indicating that elevated Zn2+ might be detrimental to neurons. However, PD research has primarily focused on investigating dopamine (DA) oxidation, resulting in the generation of reactive oxygen species that damage mitochondria and lead to neuronal cell death; there is limited research that focuses on the impact of Zn2+ homeostasis on neurodegeneration. Our previous studies have indicated that DA through the activation of D1-like receptors, induces nitric oxide synthase (NOS) to produce nitric oxide (NO), leading to increase in intracellular Zn2+ concentration ([Zn2+]i). This increment triggers autophagy, ultimately resulting in the death of primary cultured rat embryonic cortical neurons. Another common excitatory neurotransmitter, glutamate (Glu), is also known to activate NOS, but it remains unclear whether Glu increases [Zn2+]i for signaling pathway activation. Recent research has highlighted the role of inflammation as one of the factors contributing to neurodegeneration. In this study, we initially used the D1-like receptor chemically stable agonist, dihydrexidine (DHX), confirming in primary cultured rat embryonic neurons that the increase in [Zn2+]i was not due to DA oxidation but rather through D1-like receptor stimulation. Additionally, inhibition of inflammation reduced DA-Zn2+-induced neuronal cell death. Furthermore, the elevation of [Zn2+]i by DA increased the formation of NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasomes and activated downstream Caspase-1, leading to the maturation of interleukin-1 beta (IL-1β) and Gasdermin D (GSDMD). Notably, the cleaved N-terminal of GSDMD did not translocate to the cell membrane but was primarily observed in autophagosomes. Pretreatment of neurons with IL-1β reduced DA-induced neuronal cell death, suggesting that mild inflammation and its interaction with autophagy account for neuron death. Regarding Glu-stimulated cultured neurons, we observed that Glu activated NOS through ionotropic Glu receptors, leading to transient phosphorylation and subsequent synthesis of NO via the Ca2+-CaM/CaMKII pathway, causing an increase in [Zn2+]i. While this short-term increase did not impact mitochondrial activity, it elevates the expression level of NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasomes. Pretreatment with the Zn2+ chelator, TPEN, inhibited inflammasome formation. Hence, maintaining Zn2+ homeostasis is crucial in preventing inflammatory responses induced in neurons. These results demonstrate that different neurotransmitters, through their respective pathways, activate NOS to generate NO, resulting in [Zn2+]i elevation and triggering mechanisms such as autophagy and inflammatory responses, ultimately leading to cell death. After neurotransmitters activate the electrical and Ca2+ responses, Zn2+ can then be a novel signaling molecule during neurotransmission to regulate the long-term activities of neurons. Therefore, understanding how to control Zn2+ homeostasis to regulate autophagy and inflammation could offer novel strategies for treating neurodegenerative diseases. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/92277 |
DOI: | 10.6342/NTU202400419 |
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顯示於系所單位: | 生命科學系 |
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