- 學位論文
軸突核糖核酸及tau異構物在神經生長錐中的分佈隨體外發育過程變化
Distributions of axonal RNA and tau isoforms in growth cones are affected by in vitro development
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摘要
神經細胞的軸突生長錐(axonal growth cones)負責探索環境以及引導軸突(axon)往目 標物生長與移動。在此過程,軸突生長指引因子(guidance cues)會啟動局部化的蛋白質 合成並導致軸突生長錐內細胞骨架(cytoskeleton)的重組。為期瞭解生長錐中的蛋白質合 成對軸突生長所扮演的角色,我們在本研究中探討座落於軸突生長錐中的核糖核酸 (RNA)種類以及其分佈狀況。我們以限制生長錐生長之培養晶片分別收集生長錐和軸突 軸(axonal shaft)內的核糖核酸,經由定量與分類找出特別偏好位在生長錐的核糖核酸種 類。經由螢光原位雜交法(Fluorescence in situ hybridization),我們發現細胞生長條件影 響 Nefl 及 Cfl1 出現偏好位在生長錐的機率,但不影響 Cadm1 在軸突內的分佈。另外我 們也發現 Cadm1 及 Nefl 偏好位在生長錐的機會隨神經細胞成熟度增加而增加,而 Cfl1 位在生長錐的機會不變。由此研究我們認為部分核糖核酸在軸突上的分佈是受到發育及 生長環境所調控的。 我們也探討了微管相關蛋白(microtubule-associated protein) tau 的 3R 及 4R 兩種異構 物(isoform)在生長錐中的分佈隨發育成熟而改變。經由免疫螢光染色(Fluorescence immunostaining),我們發現在神經細胞體外發育早期,3R和4R tau異構物位在多個且 不特定的生長錐中。當神經細胞軸突決定之後,3R 和 4R tau 異構物便會位在軸突的生 長錐中。我們將神經細胞處理低濃度的微管穩定劑紫杉醇(taxol)發現原先座落於軸突生 長錐中的 3R-tau 異構物會減少。而將神經細胞處理低濃度的破壞微管穩定劑諾考達唑 (nocodazole),則位在軸突生長錐中的 4R-tau 異構物會減少。由此研究我們推論 3R 和 4R tau 異構物在生長錐上扮演的角色分別為與較穩定的微管及較不穩定的微管結合。
並列摘要
Axonal growth cones are the tips of axons responsible for exploring environment, growth of axons, guiding axons to move toward their targets. Local protein synthesis and cytoskeleton reorganization are rapidly executed when growth cones response to guidance cues. Here, we investigated transcripts residing in growth cones and their distributions to understand the roles played by local protein synthesis on axonal growth. We developed a chip devise for separately collecting growth cones and axonal shafts. We identified cone-preferred transcripts by quantitation and classification of transcripts found in axonal shaft and growth cones. By means of fluorescence in situ hybridization, we found that growing conditions affected cone-preferred distributions of Nefl and Cfl1 but not the distribution of Cadm1. Furthermore, we found that the fraction of cone-preferred distributions of Cadm1 and Nefl increased during in vitro development. Our results suggest the distributions of transcripts over axonal substructures were dynamical regulated by growing conditions and neuronal maturation. We also investigated the differential roles played by the two microtubule-associated protein tau isoforms, which differed in their binding ability to microtubules, in the axonal growth cones. By means of fluorescence immunostaining, we found that 3R- and 4R-tau proteins were randomly enriched in several growth cones during the early stage of in vitro development. After axon determination, we found that 3R- and 4R-tau proteins were enriched in axonal growth cones. We also found that the enrichments of 3R- and 4R-tau proteins in axonal growth cones were respectively diminished by treatment of taxol and nocodazole at low concentration. These results suggest that 3R-tau interacts with stable microtubules, while 4R-tau interacts with dynamic microtubules. Our finding implies that 3R- and 4R-tau may play differential roles in growth cones.
參考文獻
Adams, S.J., DeTure, M.A., McBride, M., Dickson, D.W., Petrucelli, L., 2010. Three repeat isoforms of tau inhibit assembly of four repeat tau filaments. PLoS One 5, e10810.
Akhmanova, A., Steinmetz, M.O., 2008. Tracking the ends: a dynamic protein network controls the fate of microtubule tips. Nat Rev Mol Cell Biol 9, 309-322.
Amos, L.A., 2011. What tubulin drugs tell us about microtubule structure and dynamics. Semin Cell Dev Biol 22, 916-926.
Andreassi, C., Zimmermann, C., Mitter, R., Fusco, S., De Vita, S., Saiardi, A., Riccio, A., 2010. An NGF-responsive element targets myo-inositol monophosphatase-1 mRNA to sympathetic neuron axons. Nat Neurosci 13, 291-301.
Aranda-Abreu, G.E., Behar, L., Chung, S., Furneaux, H., Ginzburg, I., 1999. Embryonic lethal abnormal vision-like RNA-binding proteins regulate neurite outgrowth and tau expression in PC12 cells. J Neurosci 19, 6907-6917.