檢驗NuMA和TPX2蛋白在神經突型態發生中的功能

Abstract

神經系統的功能來自於神經元之間複雜的網絡連結。為了形成正確的神經迴路，每顆神經元之間的連結必須被適當地調控，以建立有效的神經網路。神經突發生 (neuritogenesis)能確保神經正確地連結，在神經突的發展過程中佔有相當重要的地位。目前已知細胞骨架中的微管和微管相關蛋白 (microtubule-associated proteins)在神經突發生的調控中扮演了非常重要的角色。 NuMA和TPX2是兩個在細胞分裂中很關鍵的微管相關蛋白，紡錘絲的集合和維持都需要NuMA，而產生穩定的雙極紡錘絲 (bipolar spindle)則需要TPX2的參與。特別的是NuMA和TPX2不論在細胞間期或是有絲分裂後的細胞(post-mitotic cell, 例如神經元)裡都存在於細胞核中。但NuMA和TPX2在神經系統中的功能目前仍不清楚。 先前的實驗顯示以P19所分化的神經細胞為實驗材料，抑制神經細胞中的NuMA和TPX2會導致神經細胞長出較短的神經突。為了更進一步檢驗這個現象，後續實驗以小鼠海馬迴神經細胞為實驗對象，將能夠轉錄出抑制NuMA或TPX2的shRNA質體轉染進海馬迴神經細胞的初代培養 (primary culture)當中，並分析抑制NuMA和TPX2是否會改變神經突的生長。 我們發展出改良過的初代神經細胞培養方法，可培養出較健康的神經細胞，經過轉染後其存活率亦較高。本次研究中發現NuMA和TPX2確實與神經突生成相關。在小鼠海馬迴神經元中抑制NuMA或TPX2皆會降低其神經突的數量但卻不影響神經突長度。除此之外，在被抑制的神經細胞中表現外來的人類NuMA或TPX2可讓神經突數目略微回升。這些結果顯示NuMA和TPX2確實在神經突的形成(initiation)過程中扮演重要的角色。

A functional nervous system depends on the intricate connections between neurons. For neuronal circuits to be correctly wired, connections of each neuron must be properly regulated to establish an effective neural network. Neuritogenesis is an important neurite developmental process that ensures proper neuronal connections are established. It has been shown that microtubule cytoskeleton and microtubule-associated proteins are important regulators of neuritogenesis. The microtubule cytoskeleton plays an indispensable role in each of the steps during neuritogenesis. Two microtubule-associated proteins, NuMA and TPX2 play critical roles in mitosis. NuMA is an essential mitotic component to establish and maintain focused spindle pole and TPX2 is required to generate a stable bipolar spindle. Interestingly, NuMA and TPX2 localize to the nucleus during interphase or post-mitotic cells (such as neurons). However, the non-mitotic functions of NuMA and TPX2 remained elusive. A previous study showed that suppressing NuMA or TPX2 in P19-differentiated neurons resulted in shortens neurites. To confirm this effect, mouse primary hippocampal neurons are transfected with plasmids which express EGFP as a report gene and also can be transcribed into shRNA targets NuMA or TPX2. Then neurons are analyzed if NuMA or TPX2 suppressed neurite elongation. In this study, we established a novel method to culture dissociated mouse hippocampal neurons. Our method significantly simplifies the preparation while produces healthy and long-lived neuronal cultures. We also confirmed that NuMA and TPX2 indeed involve in neurite outgrowth. Suppressing NuMA or TPX2 in mouse hippocampal neurons resulted in reduced neurite number. Moreover, overexpressing human NuMA and TPX2 partially rescued the reduction of neurites. Those results suggest that both NuMA and TPX2 play important roles in neuritogenesis.