脊髓肌肉萎縮症的機制探討及治療藥物發展

To investigate the mechanism and to develop therapeutic drugs of spinal muscular atrophy

陳郁佳(Yu-Chia Chen)

指導教授 ： 鐘育志

共同指導教授 ： 游仲逸(Chung-Yee Yuo)

高雄醫學大學/醫學院/醫學研究所博士班/博士(2017年)

摘要

脊髓肌肉萎縮症(Spinal muscular atrophy, 簡稱SMA)是一種體隱性遺傳疾病，大約每一萬個新生兒中，就會有一個是脊髓肌肉萎縮症患者，為目前第二常見的體隱性遺傳疾病，同時也是造成新生兒死亡最常見的遺傳疾病。SMA是由survival motor neuron 1 (SMN1)基因突變或缺失造成，因為SMN2基因在exon 7的第6個鹼基是T(SMN1基因的該鹼基為C)，使得SMN2 mRNA在進行剪接時容易產生缺少exon 7的mRNA，導致轉譯出來的蛋白不穩定，無法彌補全長的SMN蛋白質功能。在本論文中，研究主題分為二個部分：首先，我們著重在SMN2 mRNA的剪接機制，我們發現在SMA小鼠睪丸中SMN2 mRNA剪接不同於其他組織，而是表現出較多的全長mRNA (SMN2 full-length mRNA)，我們認為在睪丸中有特定的剪接機制。為了探討在睪丸中的特定剪接機制，我們建立了SMA小鼠睪丸的初代培養細胞，我們發現睪丸細胞在經過短時間(2小時)的初代培養還是表現較多的全長mRNA，然而在經過長時間(96小時)的體外培養後則會減少全長mRNA而表現較多的缺陷mRNA (truncated form mRNA)，且在長時間培養後，細胞的Asf/Sf2及Tra2-beta1表現量明顯下降。進一步利用過度表現及剔除的技術，我們發現Tra2-beta1過度表現會促進SMN2全長mRNA的增加，而剔除Tra2-beta1則會使SMN2全長mRNA減少，證明了在SMA小鼠的睪丸中，Tra2-beta1對於調控SMN2 mRNA剪接佔有重要角色。由於目前對於SMA還沒有治癒的方法，因此對於SMA的研究，除了機制的探討，我們也致力於尋找治療方法。本論文的第二部分，就是利用藥物篩選尋找治療SMA的藥物，我們利用的是NSC34/SMN2 minigene-luciferase系統，用來篩選可以增加SMN2全長mRNA的藥物，我們發現securinine可以增加SMN2全長mRNA的表現，進一步用securinine處理SMA病人細胞也發現可以增加SMN2全長mRNA及SMN蛋白的表現。我們進一步發現securinine可以減少hnRNP A1並且增加Tra2-beta1的表現量，證明securinine透過調控這二個剪接分子影響SMN2剪接。最後我們將securinine刺激SMA小鼠，發現在SMA小鼠的腦及脊髓中，SMN2全長mRNA及蛋白的表現量也有上升的現象。這些結果證明了securinine可能成為治療SMA的藥物。在本論文中，我們對SMA疾病的研究從分子機制層面到藥物治療層面，不但有助於對SMA致病機制的了解，更致力於尋找治療SMA的藥物，希望對於SMA疾病的治療有所貢獻。

關鍵字

脊髓肌肉萎縮症 ； 選擇性剪接 ； 睪丸組織特異性 ； 一葉萩鹼

並列摘要

Spinal muscular atrophy (SMA) is an inherited neuromuscular disease caused by deletion or mutation of SMN1 gene. All SMA patients carry a nearly identical SMN2 gene, which produces low level of SMN proteins due to SMN2 mRNA exon 7 exclusion. Increasing full-length SMN protein production by promoting the exon 7 inclusion in SMN2 mRNA could be a therapeutic approach for SMA. Many studies had shown that SMN deficiency not only caused motor neuron pathology, but also leading to pathological changes across other cell types and tissues, including muscle, brain, heart, vasculature, bones, and liver. Because SMA has been known as a multi-system disorder, to investigate the mechanism of SMN2 mRNA splicing in other tissues is an important issue for SMA treatment. We found that the testis of SMA-like mice expressed high level of SMN2 full-length mRNA, indicating a specific mechanism of SMN2 mRNA splicing in the testis. We have established primary cultures of testis cells from SMA-like mice and found that primary testis cells after a 2-hour culture still expressed high level of SMN2 full-length mRNA, but the level decreased after longer cultures. We have also found that the level of Tra2-beta1 decreased during testis cell culture. Furthermore, overexpression of Tra2-beta1, increased SMN2 minigene exon 7 inclusion in primary testis cells and spinal cord neurons, whereas knockdown of Tra2-beta1 decreased SMN2 exon 7 inclusion in primary testis cells of SMA-like mice. Therefore, our results have indicated that high expression level of Tra2-beta1 is responsible for increased SMN2 exon 7 inclusion in the testis of SMA-like mice. This study has also suggested that the expression level of Tra2-beta1 may be a modifying factor of SMA disease and a potential target for SMA treatment. In addition, we screened therapeutic drugs for SMA by using SMN2 minigene-luciferase reporter system. We have found that securinine can increase SMN2 exon 7 inclusion and SMN protein expression in SMA patient lymphoid cell lines by downregulating hnRNP A1 and upregulating Tra2-beta1 expression levels. We have also found that securinine increased SMN2 exon 7 inclusion and SMN protein expressions in the brain and spinal cord of SMA-like mice. According to our results, securinine might have the potential to become a therapeutic drug for SMA disease. In this thesis, we have investigated the mechanism of SMN2 mRNA splicing in the testis of SMA mice and explored the therapeutic drugs of SMA, which provided a new issue for SMA study. The results in this thesis might contribute to SMA therapy.

並列關鍵字

Spinal muscular atrophy ； alternative splicing ； testis-specific ； Tra2-beta1 ； securinine

參考文獻

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