谷氨酸在初代培養大鼠胚胎皮質神經細胞中引發胞內鋅離子濃度上升對神經細胞死亡之影響探討

Abstract

鋅離子失衡和谷氨酸引起的興奮毒性都會促使神經細胞死亡，並造成神經退化性疾病。許多報告顯示谷氨酸會提升胞內鋅離子濃度，但機制尚未明確。我們的過去結果顯示多巴胺會透過cAMP-NO訊息路徑促使金屬硫蛋白 (MTs) 釋放鋅離子，提高胞內鋅離子濃度。在這篇研究中，我們採用鋅離子影像技術探討谷氨酸如何提升初代培養大鼠胚胎皮質神經細胞的胞內鋅離子濃度，並探討鋅離子在神經發炎中的角色。對谷氨酸受體的活化劑與拮抗劑中，NMDA能提升胞內鋅離子濃度，然而NMDA受體 (NMDAR)、AMPA受體、鈣調蛋白 (CaM)、與神經一氧化氮合成酶 (nNOS) 的對應拮抗劑，都能抑制谷氨酸引發的胞內鋅離子濃度上升。且胞外鈣離子/鋅離子螯合劑乙二胺四乙酸 (EDTA) 也降低了谷氨酸引發的胞內鋅離子濃度上升。因此，谷氨酸引發的胞內鋅離子濃度上升可能依賴於經由NMDAR流入的鈣離子及後續造成的CaM、nNOS活化。TPEN，一種鋅離子螯合劑及MCC950，一種細胞焦亡路徑的抑制劑無法抑制谷氨酸引發的神經細胞死亡，但TPEN能降低谷氨酸引發的介白素-1β，一個神經發炎相關酵素的表現上升。此外谷氨酸處理也引發ZIP2，一種鋅離子運輸蛋白的表現。這些結果解釋谷氨酸透過NMDAR-CaM-nNOS級聯來提升胞內鋅離子濃度，且此級聯對神經發炎的活化非常重要。谷氨酸作為最重要的興奮性神經傳遞物，了解其如何調控鋅離子平衡及相關基因表現將為神經退化性疾病提供新的治療策略。

Both Zn2+ dyshomeostasis and glutamate-induced excitotoxicity can lead to neuronal cell death and cause neurodegenerative disorders. Several reports have shown that glutamate stimulation can elevate the intracellular Zn2+ concentration ([Zn2+]i) but the mechanism is not clear. Our previous results have shown that dopamine elevates [Zn2+]i through the cAMP-NO signaling pathway leading to the release of Zn2+ from metallothioneins (MTs). In this study, we adopted Zn2+ imaging technique to verify how glutamate elevates [Zn2+]i in primary-cultured rat embryonic cortical neurons and examined the possibility of the activation of neuroinflammation. Glutamate and NMDA, but not AMPA or kainate (KA), could elevate [Zn2+]i; antagonists against NMDA receptor (NMDAR), AMPA receptor, calmodulin (CaM), and neuronal nitric oxide synthase (nNOS) greatly suppressed the glutamate-induced elevation of [Zn2+]i. In addition, chelating the extracellular Ca2+ and Zn2+ by ethylenediaminetetraacetic acid (EDTA) also blocked the glutamate-induced elevation of [Zn2+]i. Therefore, it is possible that glutamate-induced elevation of [Zn2+]i is dependent on the Ca2+ influx through the NMDAR and further activation of CaM and nNOS. To examine the involvement of Zn2+ and pyroptosis in glutamate-induced neuron death, we pretreated the neuron with TPEN, a cell-permeable Zn2+ chelator, and MCC950, an inhibitor of NLRP3 in pyroptosis pathway. The results showed that TPEN and MCC950 did not inhibit glutamate-induced cell death but TPEN suppressed the glutamate-enhanced expression of IL-1β. Glutamate treatment also enhanced the expression level of ZIP2, a zinc transporter protein. These results illustrate that glutamate elevates the [Zn2+]i through the NMDAR-CaM-nNOS cascade, which is important for the activation of neuroinflammation. Glutamate is the most important excitatory neurotransmitter, understanding how it regulates the Zn2+ homeostasis and related gene expression will provide new therapeutic strategy against neurodegenerative disorders.