利用果蠅感覺神經元研究樹突自我修剪過程中Ik2激酶所調控的分子機制

Abstract

神經系統的正常運作必須仰賴神經細胞之間精確地連結。當神經細胞發育時，神經樹突的多分支突起會廣泛地向外生長，以便於其他神經細胞產生聯繫，而神經細胞的樹突具有可塑性，其中樹突的自我修剪即是清除不再需要的樹突分支來達到精準的神經迴路。 果蠅的周邊感覺神經細胞，是最適合被用來觀察樹突自我修剪的模式生物，當果蠅進行完全變態的早期，其周邊感覺神經細胞會將所有的樹突分支清除乾淨，而細胞本體和軸突則是被保留下來，之後再生長出成蟲所需要的樹突分支。 在先前的研究中已知Ik2激酶是誘發樹突自我修剪中最重要的蛋白質，也找到具有螺旋區段的蛋白質Spn-F和Ik2有相互作用，並參與在樹突自我修剪的過程中。文獻回顧中指出，只有大量表現Ik2可以在幼蟲時期即誘發樹突自我修剪，因此我們想藉由研究Ik2的分子路徑，以及找出和Spn-F相互作用的蛋白質來了解樹突自我修剪的調控機制。 在本篇研究中，我們發現被Ik2所磷酸化的Spn-F必須藉由和動力蛋白(Dynein motor complex)以及Ik2形成複合體而得以散佈在神經細胞本體的細胞質中。當Spn-F沒有被磷酸化時，Spn-F會聚集成一團蛋白質聚合物在細胞質中。在果蠅進入完全變態的一小時後，Ik2的活性被蛻皮激素激發而將Spn-F磷酸化，使得Spn-F的自我蛋白質相互作用力減弱，由聚合物成為單體而散佈在細胞質中，Spn-F散佈在細胞質的過程是樹突進行自我修剪的重要調控機制。此外，我們發現小GTP酶Rab11和Spn-F有相互作用並參與在樹突自我修剪過程中。Rab11的活性對於樹突修剪非常重要，本實驗中發現，Spn-F會和沒有活性的Rab11相互作用，而Spn-F進一步能夠將Ik2和Rab11共同形成蛋白質複合物，此蛋白質複合物能夠促使Rab11的活性提高，最終使神經細胞正確地進行樹突自我修剪。 神經樹突自我修剪的過程除了參與神經細胞形成正確的神經迴路，也和退化性神經疾病的形成有相關，因此研究神經樹突自我修剪的調控機制，對於了解神經細胞的發育和神經系統疾病的發生都有很大幫助。

The precise formation of neuronal connectivity contributes to functional nervous system. During dendrite morphogenesis, nerve processes extend broadly to make connections with relevant inputs and remodel to mature neuronal circuits. The remarkable neuronal remodeling is dendrite pruning which eliminates excessive dendrites but not induces cell death. During Drosophila metamorphosis, the large-scale dendrite pruning occurs in Class IV dendritic arborization (C4da) neuron, which is the ideal model system for studying the molecular mechanisms of dendrite pruning. The previous study in our lab has revealed that IκB kinase-like 2 (Ik2), a homology of vertebrate IKKε in Drosophila, is crucial for dendrite pruning. This thesis characterized that Ik2 kinase regulates dendrite pruning through phosphorylating Spindle-F (Spn-F), the coiled-coil domain containing protein. Here, we found that Spn-F phosphorylation results in a reduced self-association and redistribution of Spn-F in soma of the C4da neuron during early metamorphosis. The redistribution of Spn-F is dependent on the functional dynein motor complex, a microtubule motor protein. While the Spn-F displays puncta pattern at larval stage, the small GTPase protein Rab11 co-localizes with Spn-F in C4da neurons. Rab11 is known to direct vesicle traffic between the endocytic and exocytic trafficking pathways. We found that Rab11 activity is required for the dendrite pruning. Spn-F has the strong affinity with GDP-bound Rab11 and links Ik2 with Rab11 to form Ik2/Spn-F/Rab11 complex. Consequently, exchange of GDP-bound Rab11 with GTP was enhanced. We revealed that elevation of Ik2 activity during early dendrite pruning induces Spn-F redistribution in a dynein complex dependent manner, and Spn-F serves as an adaptor to link Ik2 with Rab11 followed by increasing GTP-bound Rab11 which is required for dendrite pruning in Drosophila C4da neuron. It has been found that dendrite pruning occurs throughout the entire lifespan, not only involves in dendrite morphogenesis but also participates in the neurodegenerative disease. Understanding the molecular mechanism of dendrite pruning improves our knowledge of dendrite morphogenesis and progresses medical applications in neuronal disease.