Nestin-2在組織分佈及其在背根神經節發育中表現的研究

Abstract

Nestin屬於第六類中間絲蛋白質。就中間絲蛋白分子結構上來說，包含N-terminal domain、rod domain與C-terminal domain，但與其它中間絲蛋白比較不同的是，Nestin與在它的C-terminal domain會由一段11個胺基酸(S/P-L-E-E/K-E-X-Q-E-S/L-L-R)所組成的縱列重覆區域(tandem repeat)。從蛋白質分佈來說，Nestin通常只在神經上皮幹細胞以及肌肉幹細胞中表現，因此也被作為神經與肌肉發育的早期分化標誌。至於Nestin的功能，先前的報告已指出在增生細胞中，會藉由cdc2來引起在Nestin的Thr316位置磷酸化，進而促進Vimentin骨骼的組裝，以利細胞的增生。此外，Nestin也被發現具有抗細胞凋亡的功能，此功能是藉由在Thr316位址被cdk5磷酸化，並間接隔開cdk5/p35複合體，以抑制Cdk5引起細胞凋亡訊息傳遞。近來，我們實驗室發現一個全新的Nestin異構物，並命名為Nestin-2，是第一次在成體背根神經元中發現。根據序列分析發現Nestin-2缺乏縱列重覆區域，且原位雜合實驗結果也顯示，沒有任何重覆區域的mRNA可以在背根神經節被探測出來。此異構物的生成是由於選擇性剪接，而選擇性剪接不只移除重覆區域，甚至造成框架轉移，使其蛋白質C端產生一段novel pepetide。此32個胺基酸的novel peptide被用來製造一隻名為AY14(AARSRILPKEETRY)的抗體。此篇論文主旨在鑑定Nestin-2其在背根神經節發育期間之表現及各種組織的分佈狀態分別以免疫螢光染色及免疫轉漬分析進行測試。首先，收取胚胎時期(包括E11、E12、E14)以及出生後(P0、P2、 P4、P6)的背根神經節冷凍切片以AY14來進行免疫螢光染色。結果顯示胚胎時期沒有染色訊號，而P0、P2也只有微量的染色訊號，直到P4及P6才開始有比較強的免疫染色反應。另外，將背根神經節發育不同時間(包括P0, P2, P4, P6, Adult)以中間絲製備法處理後，以AY14來做免疫轉漬分析。結果顯示，在P0與P2皆沒有Nestin-2的表現，而會在P4開始表現，並且其表現量會隨著神經元的成熟而跟著上升。此外，利用AY14對中間絲製備處理的各種成體老鼠組織做免疫轉漬分析。結果顯示，除了背根神經節之外，三叉神經節、上頸神經節、脊髓以及坐骨神經都有表現，而大腦皮質以及海馬迴只有微量表現，其它組織如小腦皮質、嗅葉、骨骼肌、心肌、動脈、小腸及腎臟皆沒有Nestin-2的表現。為探究其組織內的分佈型式，以AY14來進行免疫螢光染色。發現Nestin-2在背根神經節可與表現NFH(Neurofilament H)的大直徑神經元或表現Peripherin的小直徑神經元中有co-localization的現象，特別是在那些同時表現NFH及Peripherin且中直徑大小的神經元會有較高的表現量，且在三叉神經節及上頸神經節亦顯示類似的型式。另外，在脊髓內Nestin-2的分佈主要為α運動神經元。甚者，背側神經纖維免疫螢光染色顯示出Nestin-2會與NFH或Peripherin共表現，亦會呈現Nestin-2同時與NFH及Peripherin表現的型式。而在腹側神經纖維與坐骨神經纖維中，則幾乎所有的Nestin-2皆會與NFH、Peripherin共表現。總而言之，所有結果顯示Nestin-2與Nestin有不同的表現型式，而Nestin-2只會出現於出生後的背根神經元的原因可能是由於其扮演抗凋亡的功能。 至於Nestin-2的功能，即使縱列重覆區域被移除，其Thr316的位址依然存在，可能會被Cdk5磷酸化並且在感覺神經元或α運動神經元表現出抗細胞凋亡的活性。更有甚者，Nestin-2也可表現於三叉神經元、上頸神經元、α運動神經元及神經纖維。

Nestin is one of the type VI intermediate filaments (IF) and is usually used as an early neurogenesis cell marker. Regarding the molecular structure of nestin, like other intermediate filament proteins, nestin contains an N-terminal domain, a rod domain and a C-terminal domain. However, unlike other types of intermediate filaments, the C-terminal domain of nestin contains a Tandem Repeat (TR) region. The function of nestin has been reported as facilitating the disassembly of vimentin IFs during mitosis. Furthermore, nestin also exert anti-apoptosis function through phosphorylation at Thr316 by Cdk5, which subsequently sequester Cdk5/p35 complex, thereby inhibit Cdk5-dependent apoptotic signalling. In the adult rat dorsal root ganglia (DRG), nestin is only expressed in satellite cells and Schwann cellls, but not in sensory neurons. Recently, we have identified a novel alternatively spliced isoform of nestin, named nestin-2, in adult rat DRG neurons. An isoform specific antibody, named AY14, was raised against a frameshift region specfically existed in nestin-2. We further showed that nestin-2 was expressed postnatally during DRG development. The expression of nestin-2 in the DRG neurons was first observed at postnatal day 4 (P4), and its expression level increased upon DRG maturation. In addition, the distribution pattern of nestin-2 was investigated by AY14 immunoblotting and immunofluorescence microscopy. The results showed that, in addition to DRG, nestin-2 was also expressed in trigeminal ganglia, superior cervical ganglia, spinal cord and sciatic nerves. Furthermore, multiple labeling immunofluorescence microscopy revealed that nestin-2 was partially co-localized with the neruofilament or peripherin framework. In summary, we report, for the first time, the expression of a nestin isoform that presents in mature peripheral neurons. The fact that they are expressed in postmitotic neurons indicated that they may have distinct functions rather than the full-length nestin.