Cep170之大量表現產生非中心體分佈並促使神經突生長

Abstract

微管是神經細胞組成中最重要的細胞骨架，也調控著神經細胞發育過程關鍵的角色。為了更進一步的了解微管是如何影響著神經發育，在稍早的實驗中我們利用小鼠胚胎瘤細胞來定量神經細胞分化前及分化後與微管相結合的蛋白質含量變化。其中Cep170（中心體之組成蛋白質）於神經分化後，在與微管相結合的蛋白中含量有著大量且顯著的增加，這表示著Cep170有可能在神經的微管調控中扮演著相當重要的角色。在有絲分裂細胞中，Cep170屬於中心粒的次末端附屬蛋白且有能力調控細胞中的微管分佈。然而，Cep170是否在分化後的神經元中仍參與微管的調控目前依舊是未知領域。為了解答這個問題，我們分別抑制了胚胎瘤細胞P19衍生的神經細胞及小鼠的初代海馬迴神經細胞中內源性的Cep170。意外的是，抑制Cep170對於神經分化及神經形態發生並沒有顯著的影響。相反的，在P19神經細胞及海馬迴神經細胞大量表現Cep170會造成神經突的長度有顯著的增長。為了更進一步的了解Cep170是如何促使神經突的增長，我們藉由觀察外源性的Cep170在神經中分佈的位置嘗試著判斷可能的機制。此外，我們也嘗試著利用表現不同區段的Cep170於海馬迴神經細胞中去確認Cep170促進神經突生長的蛋白質結構域。綜合以上結果，可以推測出Cep170在分化後的神經中確實扮演著相當重要的角色而且Cep170在微管上的含量與神經突的長度息息相關。

Microtubule is the major cytoskeleton in neuron, and regulates various crucial neuron developmental processes. To understand how microtubule cytoskeleton influences neuronal development, we have previously utilized quantitative proteomics to examine microtubule-associated proteomes in mouse embryonal carcinoma cells (P19 cells) before and after neuronal differentiation. One of the proteins on microtubules exhibiting substantial and significant increase upon neuronal differentiation is centrosomal protein 170 (Cep170). This suggests that it plays a role in regulating neuronal microtubules. In mitotic cells, Cep170 locates at subdistal appendages of mother centriole and has been shown to play a role in microtubule organization. However, whether Cep170 participates in microtubule organization in post-mitotic neurons remain elusive. To answer this question, we depleted endogenous Cep170 in both P19 cell-derived neurons and primary hippocampal neurons. Surprisingly, Cep170 knockdown did not significantly affect neuronal differentiation or neuronal morphogenesis. In contrast, overexpressing Cep170 caused a significant increase in neurite length in both P19 cell-derived neurons and mouse hippocampal neurons and. Furthermore, we observed that exogenously expressed Cep170 localized as puncta along neurites. We also attempted to determine the domain of Cep170 that promotes neurite elongation by overexpressing truncated Cep170 in hippocampal neurons. Our results suggest that Cep170 plays an important role in post-mitotic cells and its increase on microtubules promotes neurite formation and elongation.