以老鼠細胞株以及斑馬魚探討在肌肉中過量表現NogoA其影響運動神經元生長的機制

Using mouse cell line and zebrafish as models to study the mechanism of overexpressing NogoA in muscle which influences motor neuron growth

陳侑頡(You-Jei 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(2016年)

https://doi.org/10.6342/NTU201700038

摘要

在ALS的病人中，其肌肉內的NogoA表現量會不正常的上升，但表現過量NogoA如何造成疾病的分子機制目前仍不清楚。因此我們建立了一個可以由Doxycycline誘導，在肌肉過量表現斑馬魚Rtn4a l(哺乳類NogoA的homology)的轉殖品系Tg (pZα:TetON-Rtn4a l )。經Doxycycline的誘導一週後，可觀察到neuromuscular junction denervation的現象；三週後，肌肉束發生necrosis的現象；並且誘導四週後的成魚體重也會減輕。這些結果顯示斑馬魚肌肉中若過量表現Rtn4a l，會使其出現與人類ALS疾病相似的病變進程。
另一方面，在先前的研究中獲知，表現過量NogoA的肌肉細胞株Sol8 (Sol8-NogoA)，會減少其conditioned medium (CM)中P蛋白質的分泌量。若將運動神經細胞株NSC34培養在Sol8-NogA的CM中，其neurite的生長會受到抑制。而我們在Sol8-NogoA的CM中添加外源性的P蛋白質，發現外源性的P蛋白能夠改善因Sol8-NogoA CM所引發不利NSC34細胞其neurite生長的現象。此外，在正常肌肉細胞株(Sol8-vector)培養環境的CM，添加P蛋白的專一抗體以中和Sol8-vector CM中的P蛋白，並將NSC34培養在該溶液，發現會不利於NSC34其neurite的生長。這些結果證明P蛋白對於運動神經細胞株NSC34其neurite的生長是重要的。
更進一步，透過western blotting實驗，我們證明外源性添加的P蛋白，會使NSC34細胞內p-Limk1 S323的程度下降，進而使p-Cofilin S3的程度降低，讓神經細胞傾向於生長。但一般所知的NogoA其Nogo 66 domain下游的p-Limk1 T508的程度並不受影響。再來，我們在NSC34中分別過量表現兩種突變型的Limk1，如Limk1 T508V以及Limk1 S323A，使其與內生性的Limk1競爭，發現Limk1 T508V並不改變因P蛋白造成p-Cofilin S3程度降低的能力。反之，Limk1 S323A卻會讓外源性P蛋白促使下游p-Cofilin S3下降的能力受到影響。另外，我們以NSC34添加Nogo 66做為模擬神經生長受到抑制的平台，發現Nogo 66能使ROCK2的表現量、p-Limk1 T508、p-Limk1 S323以及下游p-Cofilin S3的程度上升，而外源性添加的P蛋白能夠透過p-Limk1 S323使Nogo 66引起的下游p-Cofilin S3上升的現象降低。同時，我們也證明Nogo 66 receptor (NgR)的抑制劑NAP2能使Nogo 66所引發的ROCK2的表現量、p-Limk1 T508、p-Limk1 S323以及下游p-Cofilin S3的程度上升的現象降低，但Pgk1卻不會使ROCK2表現量及p-Limk1 T508程度降低，說明Pgk1不影響NgR、ROCK2、p-Limk1 T508這個axis。這些結果證明：外源性P蛋白透過p-Limk1 S323位點來影響NSC34其neurite的生長是具有專一性。
最後，我們用western blotting證實Sol8-NogoA CM處理的細胞，其神經功能性markers (ChAT、Syn1)和分化傾向的markers (GAP43、MAP2)表現量都下降；但若外源性P蛋白的添加，能使ChAT、Syn1、GAP43以及MAP2的表現量都會回升。同時，細胞免疫螢光染色，也能在經P蛋白處理後的運動神經元突觸末端，偵測到較多功能性marker Syn1的訊號。總之，我們的結果證實了，NogoA在肌肉中過量表現會造成P蛋白分泌量減少，使得P蛋白影響p-Limk1 S323以及其下游p-Cofilin S3這個我們新發現的路徑的能力減弱，而造成運動神經元的病變。

關鍵字

肌肉NogoA ； 運動神經 ； 肌萎縮性脊髓側索硬化症

並列摘要

It has been reported that the protein level of muscle-specific NogoA is abnormally up-regulated in ALS patients. Since the animal model that is able to express NogoA in mature stage is less developed, we generated a zebrafish transgenic line Tg(pZα: TetON-Rtn4a l), which enables to conditionally expresses Rtn4a l in muscle after doxycycline induction. Results demonstrated that denervation at neuromuscular junction was observed after induction for one week; severe necrosis in myofibers was observed at the third week; and the weight of adult fish was greatly decreased at the fourth week. These dynamic phenotypes occurring in this transgenic line suggest that muscle-specific overexpression of Rtn4a1 in zebrafish model may exhibit similar symptoms that were suffered in human ALS disease.
The molecular mechanism underlying how over-expression of NogoA causes the ALS-like disease in zebrafish remains unclear. Previously we found that the Protein P was decreased in the condition medium (CM) obtained from culturing muscle cell line Sol8 which was over-expressed NogoA (Sol8-NogoA). In this continuous study, we demonstrated that the neurite outgrowth of neuron could also be retarded if neuron cell line NSC34 was cultured in Sol8-NogoA CM. When Protein P was directly added into Sol8-NogoA CM, the neurite outgrowth retardation occurred in NSC34 cells could be rescued. In contrast, when antiserum specifically against Protein P was added into Sol8-NogoA CM, the neurite outgrowth retardation of NSC34 remained intact, suggesting that Protein P is an essential factor for NSC 34 cells to grow neurites. Additionally, we proved that exogenous Protein P caused the decrease of the protein levels of p-Limk1 S323 and p-Cofiliin S3 in NSC34 cells; but that of p-Limk1 T508, which is the downstream of NogoA through Nogo 66 domain, remained unchanged, suggesting that the signal pathway affected by Protein P is independent of knowning Nogo 66 domain receptor (NgR1)-related pathways, including p-Limk1 T508. Furthermore, we constructed two mutated forms of Limk1 such as Limk1 T508V and S323A and transfected them to NSC34 cells. Results showed that the reduction of p-Cofilin S3 induced by Protein P was not altered by adding Limk1 T508V. Instead, the reduction of p-Cofilin S3 induced by Protein P was abolished by adding Limk1 S323A, suggesting that the normal function of endogenous Limk1 S323 in NSC34 was competed out by the mutated Limk1 S323A. We also found that addition of Nogo 66 caused the increase of ROCK2, p-Limk1 T508, p-Limk1 S323 and p-Cofilin S3. However, when Protein P was added, the levels p-Limk1 S323 and p-Cofilin S3 were decreased, suggesting that increasing p-Cofilin S3 induced by Nogo66 was ablated by Protein P through decreasing p-Limk1 S323. Meanwhile, when NAP2, a NgR1 blocking peptide, was added, the all levels of ROCK2, p-Limk1 S323, p-Limk1 T508 and p-Cofilin S3 were decreased due to no impacts of Nogo 66. Interestingly, when Protein P was added in the presence of NgR1, the levels of ROCK2 and p-Limk1 T508 did not change, suggesting that Protein P causes specifically the decrease of p-Cofilin S3 through p-Limk1 S323, which is independent of ROCK2/p-Limk1 T508 axis.
Lastly, using Western blot analysis, we demonstrated that the expression levels of neuron differentiated markers MAP2 and GAP43 and the neuron functional markers Syn1 and ChAT were decreased in NSC34 cells treated with Sol8-NogoA CM. However, if Protein P was added in the Sol8-NogoA CM, the expression levels of MAP2, GAP43, Syn1 and ChAT were all increased. The Syn1 signal was also detected in the growth cone of NSC34 using immunostaining. demonstrated that NSC34 treated with exogenous P protein exhibited more Syn1 signal at growth cone. The line of above evidence, we concluded that when NogoA was over-expressed in muscle led to decrease the secretion of Protein P, causing to reduce the ability of Protein P to decrease p-Cofilin S3 through p-Limk1 S323, resulting in inhibiting the neurite outgrowth of NSC34 cells.

並列關鍵字

NogoA ； NSC34 ； motor neuron ； ALS

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