臺灣大學: 分子醫學研究所簡正鼎王曼彧Wang, Man-YuMan-YuWang2013-03-202018-07-092013-03-202018-07-092011http://ntur.lib.ntu.edu.tw//handle/246246/247430受到許多外生環境因子與內在生理刺激的種種刺激，突觸會調整其外部形態或內在的分子機制來應對，這種突觸的可塑性對於維持其功能甚至是神經網絡的恆定是不可缺的，而近來有越來越多的研究證據顯示泛素—蛋白質降解體系統 (ubiquitin-proteasome system, UPS)在突觸的發育、重塑與維持神經迴路的穩定上扮演了重要的角色。在本篇研究裡，我們發現一個BTB-Kelch蛋白質(CG6224)對果蠅的神經肌肉鍵結發育與該突觸電生理功能的調控有很重要的影響，BTB-Kelch這一類的蛋白質是存在於泛素的E3連結酶複合體(E3 ligase complex)中、連接支架蛋白Cullin與降解標的受質的一個蛋白質橋梁，因此又稱為受質受體(substrate receptor)。 一般的軸突末端會有一些樹枝狀的分枝，在這些分枝上會有一個個澎大突起的構造，稱為varicosities或boutons，其形態就好像一個個珠子串在線上一樣，而缺乏此特定BTB-Kelch基因的果蠅突變株，其神經肌肉鍵結處的突觸型態與控制組有明顯不同：突變株的bouton數量增加很多，其中還包含了許多聚集在軸突分枝最末端的小型boutons，這些boutons除了較小之外，它們分自兩個以上的分岔(multiple branch)，此現象與一般末端的雙分岔(double-branch)不同，呼應此突變株特別的表型，我們將此特定的BTB-Kelch蛋白質命名為「很擠」 (henji)，除此之外，經由免疫螢光染色，我們分析了數種突觸的標記蛋白質(marker proteins)，結果發現後突觸緻密的一個激酶dPak (Drosophila p21-activated kinase)染色範圍在突變株裡有增大的情況，而相對的側活化區標記蛋白質FasII (FasciclinII) 訊號則減少了，電子顯微切片也可以看到在突變株的樣本中，bouton邊緣電子緻密的範圍變長，這些證據顯示在缺乏henji的情況下，果蠅神經肌肉鍵結的突觸有型態上的缺失，包含了bouton數目與外型異常、活化區範圍變大而測活化區變小以及一些地域性的蛋白質如dPak有增量的趨勢。另一方面，隨著dPak在突觸區域的量增加，受其調控的谷氨酸受體IIA (GluRIIA)的量也隨之增加，相反的，另一型的GluRIIB受體則減少，因此我們發現除了調控突觸的外部型態外，Henji也影響突觸上谷氨酸受體的組成，進而影響該突觸的電生理特性。 觀察到上述的表型，我們推測henji是經由負向調控dPak在突觸區域的蛋白量來限制該處GluRIIA/GluRIIB的比例，確實dPak的蛋白量在缺乏henji的果蠅中有全面性的增加，另外在果蠅的S2細胞中，表達Henji確實可以促進dPak受到多泛素標定(polyubiquitination)，從這些實驗我們確認了dPak是henji的降解目標蛋白。更進一步我們想知道henji突變株在神經肌肉鍵結突觸表現出的異常是否是因為dPak蛋白量過度累積所造成的，因此我們在henji突變株中減少一個基因量的dPak，發現這樣可以使GluRIIA的量回復正常，同樣的，bouton的數量與突變株相比也有部分的抑制。至於Henji是如何調控dPak，詳細的分子機制目前仍未得出結論，這也是我們未來研究的目標之一。 在這篇研究的另一部分，我們也嘗試去定義integrin betanu在果蠅神經肌肉鍵結突觸發育過程中的角色，在integrin betanu的突變株中，bouton的數量有顯著的增加，我們也描述了此受體調控bouton數量的詳細下游訊息傳遞路徑，其中包含FAK (focal adhesion kinase)與兩個受其影響的RasGAPs：(1) NF1 (neurofibromatosis1)與受其控制的cAMP路徑 (2) Vap (vascular penduncle)與受其控制的ERK-MAPK路徑，此二訊息傳遞路徑皆受integrin betanu調控並且都參與突觸發育與bouton數量的控制。In response to environmental and physiological stimulations or fluctuations, the synapse has to perform plasticity and also maintains homeostasis. Accumulating evidences support the role of the ubiquitin-proteasome system (UPS) in regulating synapse formation and remodeling, thus maintaining long-term neural circuit plasticity and homeostasis. Here we characterize the function of BTB-Kelch protein, the substrate receptor in the Cullin3 (Cul3)-organized ubiquitin E3 ligase, in the formation and neurotransmission of Drosophila neuromuscular junctions (NMJs). Mutant NMJs lacking henji activity show a dramatic increase in bouton number, including the appearance of numerous satellite boutons. Ultrastructurally, the electron-dense membrane area delineating the presynaptic active zone and the postsynaptic density (PSD) is expanded and the periactive zone is concurrently decreased in henji mutants. The PSD houses glutamate receptors (GluRs) IIA and IIB that show distinct transmissions at Drosophila NMJs. In henji mutants, the GluRIIA abundance is upregulated and the GluRIIB is downregulated. Our electrophysiological results also support a composition shift toward a higher GluRIIA/IIB ratio at henji mutant NMJs. By rescue experiments, we show that Henji acts in the postsynapse to regulate proper NMJ growth and GluRIIA/IIB composition. We further show that Henji controls NMJ growth and GluRIIA/IIB ratio by downregulation of dPak at PSDs. The postsynaptic dPAK marks the PSD area and regulates the GluRIIA abundance. Losing one copy of dpak suppressed the bouton phenotype and GluRIIA abundance in the henji mutant. Also, the intensity and area of dPAK punctates at PSDs were increased in henji mutants. We found that dPAK interacts with Henji, which promotes ubiquitination and degradation of dPak. Therefore, Henji acts at PSDs to restrict both the presynaptic bouton growth and the postsynaptic GluRIIA clustering via controlling dPAK protein level. Several questions remain to be addressed: the subcellular localization of Henji; how dPAK is regulated by Henji during its activation; and how the GluRIIA/IIB balance is controlled by Henji-regulated dPak levels. Some preliminary results and future works will be presented in the thesis. In addition, we also addressed the role of integrin betanu in restricting NMJ growth. In the loss of betanu activity, drastic increase in bouton number was observed. Further, we dissected the downstream signaling of betanu/FAK56 and reported a bifurcating cascade of NF1-regulated cAMP/PKA and Vap-mediated Ras/MAPK pathways.  5191803 bytesapplication/pdfen-US泛素-蛋白?體E3接合?神經-肌肉連結谷胺酸受體整合素betanuUPSE3 ligaseNMJglutamate receptordPakintegrin betanuthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/247430/1/ntu-100-R98448002-1.pdf