運動神經元存活蛋白於體細胞再程序化與神經分化角色之研究

Roles of SMN in Somatic Cell Reprogramming and Neuronal Differentiation in the SMA Mouse Model

林姿穎(Tzu-Ying Lin)

指導教授 ： 宋麗英

國立臺灣大學/生物資源暨農學院/生物科技研究所/碩士(2017年)

https://doi.org/10.6342/NTU201702274

摘要

運動神經元存活蛋白 (Survival motor neuron, SMN) 是一種廣泛存在於多種細胞內，並參與小核核糖核蛋白 (small nuclear ribonucleoprotein, snRNP) 與RNA剪接體 (RNA spliceosome) 生合成之重要蛋白質；此外，SMN亦參與基因轉錄轉譯調控、端粒酶 (telomerase)生合成與細胞內物質運輸等功能。SMN基因高度保留於各不同物種間，當人體因SMN1基因缺失或突變、導致SMN蛋白質功能不全，而引發脊髓性肌肉萎縮症 (spinal muscular atrophy, SMA) ，此病症為前角運動神經元 (anterior horn motor neuron) 發生漸進性退化，導致肌肉逐漸軟弱無力的一種體染色體隱性遺傳疾病。罹患此症者，通常會出現運動神經元退化、肌肉無力與萎縮等症狀；嚴重者可能出現肌張力退減、吞嚥困難甚而呼吸衰竭致死。除了其於神經細胞上扮演之重要角色外，近年研究亦指出SMN與未分化幹細胞之關聯性：SMN大量表現於果蠅之神經幹細胞與生殖幹細胞，隨著子細胞分化而呈現梯度將低；當SMN表現受抑制會造成果蠅發育障礙，過度表現則可延緩細胞分化。在哺乳動物模式中，小鼠著床前胚胎與胚胎幹細胞 (embryonic stem cells, ESCs) 皆表現大量SMN，以維持胚胎正常發育與胚胎幹細胞潛能。SMN基因剔除 (gene knockout) 具有胚致死性，使小鼠早期胚胎因細胞大量死亡而無法順利著床；此外，部分缺乏SMN之胚胎幹細胞，其維持幹細胞潛能之重要因子表現量會隨之降低、誘發細胞分化之訊息上升、後續阻礙神經分化；反之，過度表現SMN則有助於胚胎幹細胞之分化抗性。以上研究皆闡釋了SMN於細胞發育、增生與分化，以及幹細胞潛能維持之重要性，使我們提出了SMN可能影響體細胞再程序化過程之假說。誘導型多能性幹細胞 (induced pluripotent stem cells, iPSCs) 為一種近似胚胎幹細胞、具有多能分化潛力之細胞；利用過度表現特定再程序化因子 (reprogramming factor) 可將已分化之體細胞再程序化為未分化狀態。研究指出小鼠卵子中含有大量SMN，而胚胎纖維母細胞 (mouse embryonic fubroblasts, MEFs) 與分化末端之神經細胞僅表現低量SMN；基於卵細胞含有許多未知再程序化因子之前提下，更加深了SMN可能具有提升體細胞再程序化效率之潛力。本研究目的係為探討SMN於體細胞再程序化過程及神經細胞分化所扮演之角色。試驗結果顯示，SMN於體細胞再程序化過程中表現量大幅提升，並於誘導型多能性幹細胞中具有相似於胚胎幹細胞之表現量。將Smn與特定再程序化因子共同表現於體細胞，可以加速誘導型多能性幹細胞形成，同時有效增加細胞產製數量。使用SMA模式小鼠之成體纖維母細胞進行體細胞再程序化，發現低量SMN阻礙了再程序化與誘導型多能性幹細胞產製。SMA小鼠產製之誘導型多能性幹細胞中，其多能性幹細胞重要因子包含 Oct4, Sox2, Klf4, Sall4及 Rex1 之表現量皆顯著下降，由該細胞分化之成熟神經細胞數量亦減少，特定神經細胞標的蛋白如 PAX6, MAP2 及 TUJ1 表現量受抑制。此試驗結果與前述缺乏SMN表現之胚胎幹細胞相呼應。最後，我們在SMA誘導型多能性幹細胞中強制表現SMN，發現多能性幹細胞重要因子之RNA表現量隨之提升，神經細胞分化狀況亦有所改善；然而多能性幹細胞重要因子之蛋白質表現量卻呈現趨勢下降，此部分仍待進一步瞭解。綜上所述，SMN於體細胞再程序化中扮演重要角色，有助於提升SMA細胞再程序化效率與神經分化潛能。

關鍵字

運動神經元存活蛋白 ； 脊髓性肌肉萎縮症 ； 誘導型多能性幹細胞 ； 體細胞再程序化 ； 神經分化

並列摘要

Survival motor neuron (SMN) is an essential protein plays important roles in the assembly of small nuclear ribonucleoproteins (snRNPs), the components of the RNA spliceosome, and other functions include transcriptional regulation and cellular trafficking. From the aspect of evolution, SMN gene is highly conserved through diverse species and keeps fundamental functions. Deficiency of the ubiquitous SMN protein causes spinal muscular atrophy (SMA), a leading genetic cause of infant death and one of the most common autosomal recessive diseases. In addition to the roles in neuron cells, recent studies have linked the connection between SMN and stem cell potency. We and others showed that SMN regulates stem cell pluripotency, division, proliferation and differentiation in Drosophila and mouse model. On the other hand, SMN is abundantly expressed in mouse oocytes and embryonic stem cells, but low in mouse embryonic fibroblast cells (MEFs) and neurons. Therefore, we hypothesize that SMN may play important roles during the reprogramming process of induced pluripotent stem cell (iPSC) formation. The objective of this study is to examine the roles of SMN in cellular reprogramming and neuronal differentiation in normal and SMA cells. The results showed that SMN was up-regulated during the induced pluripotency reprogramming process by OSKM (Oct4, Sox2, Klf4, and cMyc) factors when ICR and C57BL/6s WT mouse MEFs were used, along with the typical pluripotency markers including Nanog, Oct4 and SSEA1. Over-expression of SMN in WT cells improved the iPSC formation efficiency as judged by real-time PCR and alkaline phosphatase (AP) activity; whereas knocking-down of SMN completely abolished the iPSC formation. Consistently, iPSC formation efficiency using SMA mouse tail-tip fibroblasts (TTFs) was extremely low, along with impaired pluripotent genes expression, including Oct4, Sox2, Klf4, Sall4 and Rex1. Reduced SMN also delayed neuronal differentiation when compared to those with normal SMN level. Finally, over-expression of SMN in SMA tail-tip fibroblasts improved the pluripotent genes expression in iPSC as well as the neurite in vitro differentiation. These findings indicate that SMN plays important roles during cellular reprogramming and neuronal differentiation.

並列關鍵字

Survival motor neuron (SMN) ； spinal muscular atrophy (SMA) ； induced pluripotent stem cell (iPSC) ； somatic cell reprogramming ； neuronal differentiation

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