肌肉的分泌性蛋白質促進運動神經元生長的分子調控機制

The Mechanism of Secreted Protein from Muscle Promotes Neurite Outgrowth of Motor Neuron

吳嘉倫(Chia-Lun Wu)

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(2019年)

https://doi.org/10.6342/NTU201900581

摘要

肌萎縮性脊髓側索硬化症（Amyotrophic lateral sclerosis，ALS ) 是一種運動神經元退化疾病，患者的神經會逐漸無法支配肌肉，造成肌肉萎縮最終無法呼吸而死亡。現今許多文獻指出，在 ALS 病患發病前，能偵測到肌肉中的 NogoA (Reticulon4, Rtn4) 過量表現，並且與疾病的產生具有正相關，不過至今為止對於其發病機制仍舊不明確。為了探討此問題，Lee 等人 (2014) 收集過量表現 NogoA 的Sol8 肌肉細胞之 conditioned medium (Sol8-NogoA CM) 處理於 NSC34 運動神經元細胞，發現突觸生長確實會受到抑制。利用二維電泳分析並比較Sol8-NogoA CM與正常Sol8 CM，找到一候選蛋白 Pgk1 在Sol8-NogoA CM中表現量下降。而Chen 等人 (2016) 外添加 Pgk1 至Sol8-NogoA CM後，則可恢復神經的生長，並使 MAP2 與 GAP43 分化 marker 以及 Syn1 與 ChAT 功能性 marker 表現量恢復、使Cofilin 磷酸化程度下降。本篇研究也發現，添加全長或是 truncated 形式 (胺基酸 314 至 417) 的 Pgk1至 NSC34，仍可降低Cofilin 磷酸化表現量。我們更進一步將 Pgk1 注射至肌肉專一過量表現 Rtn4al/NogoA 的Tg(Zα:TetON-rtn4al) 斑馬魚轉殖品系的背部肌肉與 SOD1G93A 轉殖品系 ALS 模式小鼠，皆能發現神經無法支配肌肉的情形受到延緩。為了更進一步了解 Pgk1 影響 Cofilin 磷酸化之訊息傳遞路徑，我們外添加 Pgk1 於神經細胞培養液，並針對 Cofilin可能的上游 Limk1 進行探討，發現 Pgk1 透過調控Limk1 S323而非T508位點造成鄰酸化程度下降。同時也證實Limk1 上游的Rac1-GTP、Pak1 T423、p38 T180 與 MK2 T334 的磷酸化程度也下降，說明外添加 Pgk1 至 NSC34 神經細胞會透過Rac1-GTP/Pak1-T423/p38-T180/MK2-T334/Limk1-S323/ Cofilin-S3 訊息傳遞路徑並促進突觸生長。另外，我們利用抑制細胞內能量代謝的途徑，再處理 Pgk1，發現細胞內 Cofilin 表現量仍下降，代表 Pgk1 促進神經生長之功能未經過細胞內能量代謝途徑。
綜合以上，我們發現了由肌肉分泌的 Pgk1，會影響神經細胞內Rac1-GTP/Pak1-T423/p38-T180/MK2-T334/Limk1-S323/Cofilin-S3 訊息傳遞路徑而促進神經突觸生長，並且在動物模式中Pgk1 能延緩ALS 之病徵。

關鍵字

Pgk1 ； NogoA ； 肌肉 ； 運動神經元

並列摘要

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder affecting motor neurons. The progressive degeneration of motor neurons leads to neuromuscular junction (NMJ) denervation, resulting in the loss of muscle-nerve control, which, in turn, causes muscle atrophy. Several studies have demonstrated that NogoA (Reticulon 4, Rtn4) is overexpressed in the muscle of pre-symptomatic ALS patients. However, the molecular mechanism that underlies the involvement of NogoA overexpressed in the muscle of pre-symptomatic ALS patients remains unclear. To address this question, Lee et al. (2014) collected conditional medium (CM) from cultured NogoA-overexpressed Sol8 myogenic cells (Sol8-NogoA CM) and incubated it with NSC34 motor neuron cells. Neurite outgrowth of motor neurons (NOMN) cultured in Sol8-NogoA CM was sharply inhibited. Comparison of protein profiles between Sol8-NogoA CM and Sol8 CM by two-dimensional SDS-PAGE indicated that the amount of Pgk1 was dramatically reduced in Sol8-NogoA CM compared to control Sol8 CM. However, Chen et al. (2016) discovered that when Pgk1 was added to NSC34 cells cultured in Sol8-NogoA CM, inhibition of NOMN was rescued, as were expressions of neural differentiation markers, such as MAP2 and GAP43, and functional markers, such as Syn1 and ChAT. Strikingly found in this study, with the addition of full-length, or even truncated form (amino acids 314 to 417 domain), of Pgk1, NSC34 cells exhibited a reduced expression of p-Cofilin, which is a hallmark of growth cone collapse in neuronal cells. We further injected Pgk1 directly into the dorsal muscle of transgenic zebrafish from line Tg(Zα:TetON-rtn4al), overexpressing Rtn4a1 in muscle, and hindlimb muscle of transgenic mice from line SOD1G93A, a conventional ALS model, and the results demonstrated that Pgk1 could rescue NMJ denervation. The above evidence points to secreted Pgk1 as an essential factor in the enhancement of NOMN. To understand the etiology of failed NOMN in ALS-like phenotype at the molecular level, we further studied the signaling pathway that underlies the involvement of secreted Pgk1 on the enhancement of NOMN. The results demonstrated that secreted Pgk1 reduced p-Cofilin expression by decreasing phosphorylation of Limk1 at S323, not T508 (pT508), suggesting that the well-known Pak1/Limk1 T508 axis was not involved in the signaling network of secreted Pgk1 interacted with NSC34 cells. Additionally, we found that secreted Pgk1 decreased Rac1-GTPase activity, which, in turn, reduced the phosphorylation levels of Pak1-T423, a regulator of neurological disorders, p38-T180 and mitogen-activated protein kinase-activated protein kinase 2 (MK2)-T334 within NSC34 cells. Collectively, these interactions suggest that extracellular Pgk1 secreted from myogenic cells could reduce the phosphorylation of the active Rac1-GTP/Pak1-T423/p38-T180/MK2-T334/Limk1-S323/ Cofilin-S3 axis within NSC34 cells, which, in turn, promotes NOMN. We also showed that the enhancement of NOMN within neuronal cells by extracellular Pgk1 is independent of its intracellular canonical role as an energy supplier. Overall, therefore, in the presence of NogoA-overexpressed muscle cells, the expression of secreted Pgk1 is sharply decreased, leading to various manifestations of neurodegenerative disease, including NMJ and failed NOMN, characteristic of ALS-like phenotype.

並列關鍵字

Pgk1 ； NogoA ； muscle ； motoneuron

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