運動神經元存活蛋白於小鼠胚胎發育與幹細胞潛能角色之研究

Abstract

運動神經元存活蛋白（Survival motor neuron, SMN）是一種參與small nuclear ribonucleoproteins (snRNPs)組裝的蛋白質,並作用於細胞轉錄調控和胞內運輸功能。做為跨越生物界的高度保留基因, SMN1突變會造成脊髓性肌肉萎縮症(spinal muscular atrophy, SMA)。該病症屬自體染色體隱性遺傳疾病，發生機率為1/6000~1/10000，在臺灣每年約有25個新生兒罹患此症。罹患此症之病人，通常會出現運動神經元萎縮與肌肉的退化，最嚴重者甚或導致死亡。近期研究指出，SMA之決定因子SMN會大量表現在果蠅的幹細胞中，且隨著分化之進展而呈現梯度地降低，在分化末端之細胞僅表現少量的SMN。依據該模式，SMN發生缺陷除抑制幹細胞之生長外，亦將導致幹細胞之快速分化，顯示SMN於細胞發育、增生與分化扮演重要角色；惟此一新發現目前迄未在哺乳類幹細胞中被證實。本研究目的係有系統地探討SMN在小鼠早期胚發育過程及後續胚胎幹細胞(embryonic stem cells, ESCs)潛能中所扮演的角色。試驗結果證明，SMN在著床前囊胚之內細胞群、快速分裂之胚胎幹細胞及生殖腺細胞中大量表現。若經由人工產生的短片段干擾核醣核酸降低胚胎幹細胞中SMN基因表現量，多能性幹細胞的重要因子如Sox2, Klf4, 及Sall4等之表現量皆隨之下降，且有助於開啟誘導分化之細胞訊息傳遞，如：ERK pathway。此等結果呼應了SMN在果蠅幹細胞中梯度表現與分化的關係。反之，缺乏SMN的胚胎幹細胞，除了無法順利表現神經幹細胞之標的蛋白PAX6與NESTIN外，亦影響後續神經細胞的成熟過程；若將SMN強制表現於胚胎幹細胞時，能有效抵抗Retinoid acid所誘導之分化，並產生更多神經細胞與增加神經的長度。在小鼠的生殖腺細胞中， SMN亦大量表達於性成熟公鼠之快速生長之精母細胞與母鼠發育中之卵子。此外，在精原幹細胞中SMN會特定表現於少數細胞，顯示SMN與細胞增生確有重要之關聯性。進一步使用SMA模式小鼠，發現此種小鼠之性腺組織的生長減慢，精原幹細胞數量明顯減少，並且在母鼠卵巢也有顯著之凋亡閉鎖濾泡產生。綜合上述試驗結果證明SMN在小鼠胚胎發育過程、胚胎幹細胞潛能、神經細胞分化與生殖細胞生長中皆扮演了重要的角色。

Survival motor neuron (SMN) is a protein involved in the functions in assembly of snRNPs, transcriptional regulation and cellular trafficking. SMN gene is highly conserved across widely diverse species over biological kingdoms. Mutations in the SMN1 gene cause spinal muscular atrophy (SMA), an autosomal recessive disease and the most common genetic cause of childhood mortality. The incidence of SMA is approximately 1/6000-1/10,000 every live birth, and there are about 25 affected infants every year in Taiwan. In the most severe type, the consequences of SMA often reveal motor neuron loss, muscle degeneration and death. In a Drosophila model, it has been found that SMN expresses abundantly in stem cells and reaches its lowest level in differentiated cells. Deficiency of SMN leads to growth delay and rapid differentiation, indicating that SMN correlates to stem cell division, proliferation and differentiation. However, it is unclear whether a similar effect exists in vertebrates. Using mouse model, the results show that SMN is enriched in the inner cell mass of late blastocyst, embryonic stem cells (ESCs) and germline cells. Reduction of SMN impairs the pluripotent gene expression in ESCs, such as Sox2, Klf4 and Sall4. Moreover, it is found that the reduction of SMN activates ERK signaling and affects neuronal differentiation in vitro. Reduced SMN delays the cell growth and shows weaker signals of neuronal stem cell markers, like PAX6 and NESTIN, compared to those with a normal level of SMN. Over-expression of SMN provides protective effect for ESCs from retinoic acid-induced differentiation and stimulates neurite formation in in vitro differentiation. On the other hand, SMN also expresses abundantly in mouse germline cells, particularly in spermatocyte and growing oocyte. SMN is enriched in some spermatogonia stem cells, implying the loss of SMN might affect stem cell growth. In SMA model mice, the growth of gonadal tissues is decreased and the expression of spermatogonium marker is also down-regulated. More atretic follicles appear in the ovaries of female SMA mice. Taken together, our results suggest that SMN plays a role in the maintenance of stem cells, including pluripotent ESCs, neuronal progenitor, and male germline stem cells in mice.