碳酸鋰對過度表達α-Internexin的PC12神經細胞 存活的保護作用

Abstract

神經纖維蛋白 (neurofilament) 在神經細胞中的異常堆積，是神經退化疾病中常見的致病原因，而治療方法仍尚未明朗。為了在體外模擬神經纖維蛋白異常堆積的環境，我們實驗室先前的研究中建立了中間絲蛋白(α-Internexin)過度表達的PC12細胞株 (pINT-EGFP PC12)，並在以神經生長因子 (nerve growth factor, NGF) 誘導分化之後，發現細胞株在α-Internexin異常堆積之下最終會邁向細胞凋亡。本實驗室過去的研究中發現，能使神經纖維蛋白磷酸化的特定激酶Cdk5和GSK-3β若被抑制，則會減緩細胞中的神經纖維堆積，從而避免細胞胞器的損傷，達成延緩細胞死亡的效果。本研究將以常見的神經疾病藥物-碳酸鋰 (Lithium carbonate) 來作為抑制劑，來確認其對於神經保護以及延緩細胞死亡的功效。 在本論文中，我們主要目的是以Lithium carbonate對誘導分化後的pINT-EGFP PC12細胞株進行處理，並觀察神經纖維蛋白質層面以及細胞的形態上的變化，最後測試其延緩細胞死亡的功效。pINT-EGFP PC12細胞在NGF誘導分化後的第四天，給予含有Lithium carbonate的條件培養基。經過培養二到六天，利用西方墨點法 (Western blot assay) 、細胞免疫染色 (Immunocytochemistry) 和斷裂 DNA 分析 (TUNEL assay) 對細胞進行分析、觀察。已分化pINT-EGFP PC12細胞株經由Lithium carbonate處理後，在Western blot中觀察到神經絲蛋白重鏈 (neurofilament heavy chain, NF-H) 的磷酸化比率有下降的趨勢。而在細胞免疫染色實驗中，可觀察到神經絲蛋白重鏈、中鏈(neurofilament medium chain, NF-M) 及輕鏈 (neurofilament medium chain, NF-M) 有較高強度的螢光表現在pINT-EGFP PC12細胞突起之中。TUNEL assay結果顯示，在對照組和鋰處理組中都可以檢測到細胞凋亡信號。然而，相較於未以抑制劑處理的對照組，以Lithium carbonate處理的組別，其細胞死亡率趨勢較為平緩。 本研究證明了Lithium carbonate可以延緩分化後的pINT-EGFP PC12細胞的死亡，但是也證明Lithium carbonate能夠抑制神經纖維蛋白的磷酸化，並在誘導分化後的pINT-EGFP PC12細胞中幫助神經纖維蛋白維持其功能性。因此Lithium carbonate將可能作為研究與過度磷酸化細胞骨架相關神經退行性疾病的潛在藥物。

Accumulation of hyper-phosphorylated cytoskeletal proteins in neurons is a common pathogenic factor in the neurodegenerative diseases. To model this in vitro, a PC12 cell line overexpressing α-internexin (pINT-EGFP PC12) has been established to mimic the neuronal degenerative pattern with accumulation of hyper-phosphorylated cytoskeletal proteins. After neuronal differentiation induced by the nerve growth factor (NGF), these PC12 cells exhibited apoptosis due to abnormal neuronal intermediate filament accumulation. Prior research indicated that inhibiting specific kinases, cyclin-dependent kinase (Cdk5) and glycogen synthase kinase-3 beta (GSK-3β), which phosphorylate neurofilament (NF) proteins, could reduce cytoskeletal accumulation in cytoplasma and prevent organelle damage, thereby delaying cell death. In present study of PC12 cell models, we evaluated the possible neuroprotective effects of lithium carbonate, a common drug for bipolar disorder, might as well to inhibit the phosphorylation of cytoskeletal proteins. In this study, we aimed to assess the impact of lithium carbonate on differentiated pINT-EGFP PC12 cells, focusing on NF protein levels and cell morphology, and its potential to delay apoptosis. pINT-EGFP PC12 cells were treated with lithium carbonate-containing medium from the fourth day of NGF-induced differentiation. Over a treatment period of two to six days, we conducted analyses using Western blot, immunocytochemistry, and TUNEL assay. Post-treatment, a decrease of phosphorylation in NF heavy chain (NF-H) was observed in Western blot analyses. Besides, the higher immunofluorescence intensity of NF-H, NF-M, and NF-L could be found in the neurites of pINT-EGFP PC12 cells by immunocytochemistry. Apoptotic signals were detected in both control and lithium-treated groups via the TUNEL assay. However, the trend in the cell for the lithium-treated groups was more gradual compared to the untreated control groups. This study demonstrated that lithium carbonate could delay the cell death in differentiated pINT-EGFP PC12 cells. It also suggested that lithium carbonate inhibited NF phosphorylation and might help to maintain neuronal function. It is suggested that lithium carbonate could be a potential candidate for further studies in hyperphosphorylated cytoskeleton-related neurodegenerative diseases.