研究驅動蛋白KIF21B在神經突發生所扮演的角色

Abstract

一個有功能的神經系統是架構在神經元之間適當的連結上。神經突發生(neuritogenesis)是神經突(neurites)發展的過程，對於神經突的正確連結有著關鍵性的角色。先前的研究指出一些在微管上的蛋白microtubule-based motors在神經突發生的過程中有許多的功能，像是胞內運輸、微管之間的運動等等。這些microtubule-based motors分解ATP並以其化學能在微管(microtubule)上做單方向的運動，並可分做dynein superfamilies和kinesin superfamilies。 KIF21B是kinesin superfamilies的一員，先前的報導指出它是第一個被發現有樹突專一性的kinesin。但是對於它在神經細胞的功能的研究一直都不多。先前實驗室的研究發現，在類幹細胞P19分化為神經後，微管中KIF21B的含量有顯著的增加。這暗示著它可能在神經的分化或發展過程中扮演重要的角色。 在這份研究中，我們利用免疫螢光染色(immunofluorescence)和RT-PCR (reverse transcription PCR)驗證了P19細胞分化前後KIF21B的表現量顯著增加。利用免疫螢光染色也驗證了KIF21B在初級海馬迴神經細胞(primary hippocampal neurons)中主要存在於神經細胞本體(soma)和樹突中。藉由shRNA在P19衍生神經和初級海馬迴神經細胞中進行專一性的knockdown，我們發現抑制KIF21B在神經突發生的初級步驟中並無明顯的影響。為了要能夠更有效率的轉殖初級海馬迴神經，我們也製造出高感染能力且低細胞傷害性的慢病毒轉殖系統以利未來用在組織轉殖和活體動物轉殖上。

A functional nervous system is established on the proper neurite connections that form between neurons. Neuritogenesis is a neurite developmental process and crucial in forming proper neuronal connections. Previous reports had shown that microtubule-based motors play important roles in multiple steps in neuritogenesis. They can be divided into two superfamilies, dynein superfamilies and kinesin superfamilies (KIFs). KIF21B is a member of the kinesin superfamily and has been proposed to be a dendrite-specific kinesin. But the function of KIF21B in neurons remains elusive. In previous study using P19 system, the percentage of KIF21B on MTs increased significantly upon neuronal differentiation. This implicated that it might play a role in neuritogenesis. In this study, the increase in the expression level of KIF21B during P19 neuronal differentiation had been confirmed by immunofluorescence (IF) staining and reverse transcription PCR (RT-PCR). Using IF staining in dissociated primary hippocampal neurons, I confirmed that KIF21B localized mainly in the soma and neurites, and preferentially in the cytoplasm. KIF21B distributed into all neurites but was enriched in dendrites. Through shRNA-mediated knockdown in P19-derived neurons and dissociated hippocampal neurons, I found that KIF21B depletion produced no observable phenotypes during early neuritogenesis. In order to transfect hippocampal neurons effectively, the lentivirus production system were established.