MK-801 抑制GT1-7 細胞中細胞色素氧化
酶次單位II 表現的機制

本實驗室最近的研究結果指出雄鼠在性別發育的過程中 SDN-POA 核區NMDA 受器表現量較多，對於保護神經元免於凋亡扮演著一個重要的角色。以MK-801 阻斷後，發現neurotrophic (RBM3, P19, Cytoskeletal gamma-actin, alpha-tubulin)基因及與細胞凋亡相關基因(Bcl-2, cytochrome oxidase subunit II, cytochrome oxidase subunit III, Cytochrome b)表現明顯減少。有趣的是，Cytochrome oxidase subunit II (COII)，是由粒線體內基因所表現。當其表現明顯減少時，可能造成ATP 損耗而引起細胞凋亡。然而，NMDA 受器活化後如何影響粒線體DNA 轉錄作用的訊號傳遞路徑目前還不清楚。但是目前已有文獻指出，NMDA 受器的活化作用，造成鈣離子流入細胞內可能會活化PGC-1，並增加NRF-2 與mtTFA 的結合能力，而mtTFA對於COII 的轉錄作用是不可或缺的。因此，本實驗設計為了瞭解 NMDA 受器調控神經元存活的過程，COII 所扮演的角色。我們利用 GT1-7 細胞來探討NMDA 受器調控粒線體基因的訊息傳遞。研究中以MK-801 阻斷NMDA 受器後，以流式細胞儀(Flow cytometry)偵測細胞內鈣離子的濃度；以RT-PCR 及Western blot analysis 來觀察 MK-801 處理後PGC-1 的表現，COII 的表現。另外，以electro-mobility shift assay (EMSA)觀察NRF-2 與DNA 的結合活性，最後，以TUNEL染色觀察是否有細胞凋亡。結果顯示在GT1-7 細胞處理MK-801 後會造成(1) 細胞內鈣離子的不足，發生於20 小時後；(2) 24 小時後， PGC-1 mRNA 及蛋白質的表現明顯減少；(3) COII mRNA 的表現減少發生於36 小時；(4) GT1-7 細胞凋亡則於48 小時後顯著增加。因此，阻斷NMDA 受器可能依序導致細胞內鈣離子缺乏，PGC-1 減少，COII 受抑制，並引起後續神經細胞凋亡。

關鍵字

鈣離子；細胞色素氧化?次單位II

並列摘要

The predominant expression of NMDA receptor in the neurons of SDN-POA (sexual dimorphic nucleus of preoptic area) of male rats plays an important role in preventing neurons from apoptosis during sexual development. Blockade of NMDA receptor by antagonist (MK-801) downregulates some apoptosis-related genes, including Bcl-2, cytochrome oxidase subunit II, cytochrome oxidase III, and cytochrome b. Among them, interestingly the reduced expression of cytochrome oxidase subunit II (COII), a mitochondria-encoded complex IV subunit, may lead to ATP depletion and apoptosis. However, the down-stream signaling pathway of NMDA receptor activation to affect mitochondrial DNA transcription remains unclear. Since activation of NMDA receptor causes calcium influx and may increase the activities of PGC-1 and NRF-2 to transactivate mtTFA, which is essential for transcription of COII. This study aims to clarify the mechanism by which blockade of NMDA receptor down-regulates COII gene expression. GT1-7 cell was used as a model system. Intracellular calcium concentration ([Ca2+]i) was detected by flow cytometry using Fluo-3 AM. The mRNA expression and protein content of PGC-1 were compared between control and MK-801-treated cells by RT-PCR and Western blot analysis, respectively. The DNA binding activity of NRF-2 was evaluated by electro-mobility shift assay (EMSA). COII gene expression after the blockade of NMDA receptor by antagonist (MK-801) was detected by RT-PCR. And apoptotic cell death was shown identified by TUNEL (Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-endlabeling) staining. Our results showed that, after MK-801 treatment in GT1-7 cells, (1) The calcium deficiency occurred after 20hrs. (2) The expression of PGC-1 mRNA and protein were significantly suppressed after 24hrs. (3) COII mRNA was down-regulated after 36hrs. (4) Neuronal apoptosis was induced after 48hrs. Therefore, blockage of NMDA receptor may sequentially cause intracellular insufficiency, PGC-1 inhibition and COII expression, which may lead to subsequent neuronal apoptosis.