以小鼠細胞株以及斑馬魚研究細胞外Pgk1與其神經膜受器的交互作用會促進運動神經突的生長

Using Mouse Cell Line and Zebrafish as Models to Determine that the Neurite Outgrowth of Motor Neurons is Enhanced by the Interaction between Extracellular Pgk1 and its Neural Receptor

陳泓宇(Hong-Yu Chen)

指導教授 ： 蔡懷楨

國立臺灣大學/生命科學院/分子與細胞生物學研究所/碩士(2022年)

https://doi.org/10.6342/NTU202203910

摘要

肌萎縮性脊髓側索硬化症 (Amyotrophic lateral sclerosis, ALS) 是由於脊髓的運動神經元退化而逐漸無法支配肌肉，病人肌肉逐漸萎縮而癱瘓，最終由於橫膈膜肌肉也逐漸萎縮，導致病人無法呼吸而死亡。近年來發現extracellular Phosphoglycerate Kinase 1 (ePgk1)會透過Rac1-GTP/Pak1-T423/p38-T180/MK2-T334/Limk1-S323/ Cofilin-S3 訊息傳遞路徑來促進運動神經元的神經突生長。然而，尚不清楚骨骼肌肉細胞所分泌的 ePgk1 如何觸發運動神經元細胞，以經此傳遞路徑增強它們的神經突生長。另一方面，最近又發現若把重組Pgk1浸泡在NSC34神經細胞培養液發現細胞膜周圍可以檢測到外加的重組Pgk1信號，且透過Pull down/LC-MS/MS分析可能與Pgk1結合的神經細胞membrane proteins，接著利用Cell-surface Cross-linking immunoprecipitation找到神經細胞膜蛋白Py2與分泌的ePgk1有著最高的親和性。所以，本研究想進一步確認運動神經元膜上存在著Py2 receptor，它會與肌肉細胞分泌的 ePgk1 ligand相互作用以觸發信號傳遞，而增強運動神經元的神經突生長。首先，我在NSC34細胞中過量表現Py2，發現Py2分佈於細胞膜上，且在其培養液中添加Pgk1則會在細胞膜上與Py2產生信號重疊。其次將 py2過量表現於Tg(mnx 1:GFP) 斑馬魚轉殖品系，透過觀察胚胎之初級運動神經元 (primary motor neurons) 發現過量表現py2會增強促進神經突生長的能力；若過量表現py2再浸泡Pgk1則對其神經突生長的促進力具有加乘性的作用。相反地，若將py2 knockdown時，則會使其神經突生長被阻礙。另外，當注射truncated forms的 py2，那促進神經突生長的能力便會消失，表示ePgk1 domanin 促進神經突生長是重要的。最後，我應用分子對接 (molecular Docking) 程式推測py2與Pgk1 透過電荷(Electrostatics)作用將兩蛋白質以ligand和 receptor方式結合。有趣的是當我用突變Pgk1浸泡斑馬魚胚胎時，那促進神經突生長的能力就會消失。綜合以上，我們利用in vitro system的NSC34細胞與in vivo system的斑馬魚轉基因品系Tg(mnx 1:GFP)證實了py2作為運動神經細胞的membrane receptor，與ePgk1的ligand產生結合而傳遞訊息，以促使細胞斑馬魚胚胎運動神經元的神經突生長。

關鍵字

斑馬魚 ； 小鼠細胞株 ； 磷酸甘油酸激酶 ； 膜受器 ； 運動神經突

並列摘要

Amyotrophic lateral sclerosis (ALS) is the progressive loss of muscle innervation due to the degeneration of motor neurons in the spinal cord, resulting in gradual atrophy and paralysis of muscles. Our lab previously found that extracellular Phosphoglycerate Kinase 1 (ePgk1) secreted from skeletal muscle cells could promote the neurite outgrowth of motor neurons through the Rac1-GTP/Pak1-T423/p38-T180/MK2-T334/Limk1-S323/ Cofilin-S3 signaling pathway. However, how ePgk1 could trigger motor neuron cells to promote neurite outgrowth via this pathway remains unknown. More recently, our lab also reported that when recombinant Pgk1 was added into the medium cultured mouse motor neuron hybrid NSC34 cells, the signal of recombinant ePgk1 could be detected around the cell membrane. Furthermore, when we used pull down/LC-MS/MS combined with cell-surface cross-linking immunoprecipitation, one of neural cell membrane proteins, Py2, showed the highest affinity with ePgk1. Therefore, in this study, I proposed that Py2 might serve as a receptor to interact with the ligand ePgk1 and enhance the neurite outgrowth of motor neurons through triggering the signal transduction described above. To answer this issue, first, I overexpressed Py2 cDNA in NSC34 cells and observed that Py2 was distributed around the cell membrane, which was colocalized the signal from the ePgk1 added in the culture medium. Second, I microinjected py2 mRNA into the transgenic zebrafish line Tg (mnx 1:GFP) and found that the neurite outgrowth of primary motor neurons was increased in embryos. Moreover, if overexpression of py2 mRNA combined with supplementary addition of ePgk1, I found that the neurite growth of motor neurons was increased synergistically. In contrast, knockdown of py2 in embryos displayed the retarded growth of motor neurons. Additionally, the capability of neurite outgrowth promotion was lost if I overexpressed the truncated form of Py2, suggesting that the domain is important for ePgk1 in the promotion of neurite outgrowth. Finally, using molecular docking program, I predicted that Py2 receptor might interact electrostatically with ePgk1 ligand. Interestingly, the promotion of neurite outgrowth was failure in the zebrafish embryos incubated with the mutant Pgk1, suggesting that the fragment within Pgk1 is a critical structure for ePgk1 function. Taken together, I concluded that Py2, serving as a membrane receptor of motor neurons, interacts with the ligand of ePgk1 to trigger the molecular signal, resulting in promotion of neurite outgrowth of zebrafish embryonic motor neurons.

並列關鍵字

zebrafish ； mouse cell line ； Pgk1 ； membrane receptor ； motor neurite

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