- 學位論文
背根神經元巢蛋白異構物Nes-S：辨識、鑑定與細胞保護功能
Identification, Characterization and Cytoprotective Function of a Novel Nestin Isoform, Nes-S, in Dorsal Root Ganglia Neurons
摘要
巢蛋白 nestin 是一種第六類中間絲蛋白，專一性地表現於神經及肌肉幹細胞中。Nestin 幾乎從未在成熟神經元中被發現過，此外，它的異構物也從未被報導過。在本研究中，我們利用單神經元 RT-PCR 技術，自成熟大鼠背根神經元中，鑑定第一個 nestin 異構物— Nes-S。在此異構物中，原本 nestin 蛋白絕大部份的C端區域，包括整個重覆片段區域 ( repeat region )，皆因選擇性剪切 ( alternative splicing ) 而移除。該剪切亦同時造成了下游序列的框移突變 ( frameshift mutation )，導致切點下游產生了一段長32胺基酸 (aa)的全新片段，以及讓轉譯提前結束的停止密碼子 ( premature stop codon )。Nes-S 蛋白總長度為 408-aa，其中包含 7-aa 長的 N 端區域、一個完整、307-aa 長的 Rod domain，以及一段 94-aa 長的 C 端區域，其預測的分子量為 45.9kD。在不表現胞質中間絲蛋白的 SW13 細胞株中，Nes-S 無法自我組裝形成中間絲纖維。取而代之的是，Nes-S 在表現 vimentin 的 SW13 細胞中，能夠與 vimentin 共同組裝形成中間絲纖維。在 N2a 神經母細胞瘤細胞株、以及初代培養的背根神經元中，Nes-S 亦可和 peripherin ，或是 neurofilament heavy chain 共同形成中間絲纖維。為了研究 Nes-S 的表現分佈狀況，我們從其尾端的變異序列區域，挑選並合成出適合的多胜片段，並製造專一辨識該片段的兔多株抗體。利用此抗體進行免疫轉漬法實驗，我們發現，直至大鼠出生後第五天，Nes-S 始於背根神經節中表現，且該表現會一直持續存在至成體。此外，利用同一支抗體進行免疫轉漬實驗和免疫標記實驗發現，在我們檢視的組織當中，Nes-S 僅會表現於會將其神經延伸至身體周邊或四肢的神經元中，其中包括三叉神經節和 superior cervical ganglion 之神經元，以及胸椎脊髓中的交感神經元和運動神經元。最後，透過 MTT assay，我們發現當 Nes-S 被轉殖於 N2a 細胞中時，將減緩遭受氧化壓力下的 N2a 細胞自戕反應。此外，若將初代培養之成鼠背根神經元中既有的 Nes-S 以 RNAi 敲減，將使該細胞喪失於長期培養環境中存活的能力。過去十年以來，越來越多證據指出基因選擇性剪接是另一種調控中間絲蛋白功能的方式。而這些中間絲蛋白異構物亦可能進一步參與細胞壓力反應路徑，甚至參與神經退化性疾病的進程。Nes-S 是一種新的、能在成熟神經元中行使保護作用的中間絲蛋白，未來專注于 Nes-S 上的研究，將可能有機會為神經退化性疾病研究領域，產生新的貢獻。
並列摘要
Nestin is a type VI intermediate filament (IF) protein predominantly expressed in neurogenic and myogenic stem cells. Nestin has hardly been discovered in mature neurons, and its isoforms have never been reported. In this study, the first nestin isoform, Nes-S, was identified by RT-PCR on RNA obtained from single neurons of dorsal root ganglia (DRG) of adult rats. In this isoform, the majority of the nestin C-terminal domain, including the whole repeat region, is removed. This splicing also changes the open reading frame, and results in a novel 32-aa tail sequence and a premature stop codon. The Nes-S protein is 408-amino acid (aa) in length, containing a complete 7-aa N-terminal domain, a complete 307-aa rod domain, and a 94-aa C-terminal domain, with a predicted molecular weight of 45.9 kD. Nes-S cannot form filaments by itself in cytoplasmic IF-free SW13 cells. Instead, it co-assembles into filaments with vimentin in vimentin+ SW13 cells, and with peripherin and neurofilament heavy chain in both N2a neuroblastoma cells and primarily cultivated DRG neurons. To study its distribution pattern, an isoform specific antibody was generated against a peptide derived from the novel tail sequence of Nes-S. Immunoblotting assay with this antibody showed that its expression first appears in DRG at postnatal day 5 and persist to adulthood. In addition, immunoblotting and immunofluorescence microscopy with the same antibody showed that, among the adult neural tissues we examined, expression of Nes-S was only observed in neurons whose processes extend towards the limbs or the periphery of body, including neurons of DRG, trigeminal ganglia, superior cervical ganglia, as well as motor and sympathetic neurons of the thoracic spinal cord. Finally, we showed by MTT assay that Nes-S exerts a cytoprotective function when transfected into N2a neuroblastoma cells, and knockdown of endogenous Nes-S impaired the survival of primary DRG neurons during prolonged cultures. For the last decade, emerging evidences have revealed that alternative splicing serves as an additional way to regulate IF functions, and the IF isoforms may be involved in cellular stress responses as well as the pathogenesis of neural degenerative diseases. Nes-S is a new neuronal IF protein that exerts a cytoprotective function in mature neurons, and future studies on this protein may shed new lights on the research of neural degenerative diseases.
參考文獻
延伸閱讀
- Cheng, H. H. (2007). 神經元樹突小刺細胞質中蛋白質動態分佈機制之研究 [doctoral dissertation, National Tsing Hua University]. Airiti Library. https://doi.org/10.6843/NTHU.2007.00461
- Li, Y. C. (2014). 過度表達紅血球生成素的 NIH/3T3 纖維母細胞株之神經元保護作用研究 [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU.2014.02607
- Tseng, C. W. (2015). 應用主成分分析於老鼠胚胎幹細胞免疫沈澱定序資料揭示轉錄因子與組蛋白修飾標記在特定區域的顯著關係 [master's thesis, National Chiao Tung University]. Airiti Library. https://doi.org/10.6842/NCTU.2015.00112
- Wei, J. J. (2012). Significant pathways and functional modules by differential PPI network in three hESCs differentiation processes for the development of DA neurons [master's thesis, National Tsing Hua University]. Airiti Library. https://doi.org/10.6843/NTHU.2012.00623
- Huang, Y. L. (2009). The study of expression, regulation and function of an intermediate filament protein nestin in rat vascular smooth muscle cells [doctoral dissertation, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU.2009.00228