Henji與蛋白酶體於果蠅肌肉神經連結調控IIA型谷氨酸受體量的機制探討

Abstract

為因應環境與生理變化，神經突觸會調整可塑性並同時維持恆定性，細胞內的蛋白質組須受到嚴密的調控以應對這些挑戰，蛋白質新生成、運輸以及降解摺疊錯誤與受損的蛋白質在在是維持胞內蛋白質組的重要環節。 第一部分中分析了henji(亦稱dbo)之基因突變株於果蠅肌肉神經連結突觸的性狀，在此突變株中，富含突觸的神經小結(boutons)出現異常的型態：許多小型的聚集在分支最末端的小結，稱為衛星狀小結 (satellite boutons)。另一方面，穿透式電子顯微鏡的超微結構分析顯示後突觸緻密(postsynaptic density, PSD)的區域增大，在後突觸緻密中聚集著許多後突觸特有的蛋白質以及谷氨酸受體GluRIIA與GluRIIB，這兩類的谷氨酸受體具有不同的離子通道特性，因此一個後突觸包含的谷氨酸受體組成(亦即GluRIIA與GluRIIB的比例)大大影響了該突觸的電生理反應。在henji突變株中GluRIIA異常的大量累積在突觸處，而GluRIIB並無明顯變化，電生理測量也顯示出突變株的突觸具有較高的GluRIIA/GluRIIB比例。同樣在henji突變株中也發現dPAK這個已知可促進突觸處GluRIIA累積的激酶也增加了，當henji突變株中dpak的基因量減少一半時，GluRIIA的量與衛星小結的數量都恢復正常，另外我們也證明Henji透過其Kelch domain與dPAK產生分子間的作用，並且Kelch domain對於Henji在細胞內的位置以及調控GluRIIA的功能都是不可或缺的，因此我們的結論是Henji在後突觸透過調控dPAK來控制GluRIIA在突觸處的量以及前突觸小結的正常生長。 第二部分中首先分析了蛋白酶體在果蠅神經肌肉連結的分布位置，發現突觸下質網(subsynaptic reticulum, SSR)的區域缺乏蛋白酶體，而肌肉細胞質的其他區域都有蛋白酶體的分布，此缺乏蛋白酶體的「冷區」並非由於SSR的物理屏蔽阻隔而形成的，而是蛋白酶體藉由Ecm29這個連結蛋白固定在SSR區域外面。當再後突觸以RNAi的方式來抑制蛋白酶體的功能時，GluRIIA在突觸處的量快速累積而GluRIIB則無明顯變化，藉此來快速調整GluRIIA在突觸處的量可使整個後突觸得以補償突然的神經元活動變化，確實在短暫抑制神經元活性時，蛋白酶體錯位跑到SSR區域內，並且GluRIIA在突觸處的量急速增加而GluRIIB則沒有變化，我們猜測即時增加GluRIIA的量可以提高對突觸前刺激的反應，或許可以補償神經元活性的降低。在抑制神經元活性的情況下增加胞內cAMP/PKA的活性，可以部分恢復蛋白酶體的錯位，使其重新聚集在SSR外面。由上述實驗結果我們認為蛋白酶體經由Ecm29固定在SSR區域外圍，藉此減少SSR區域內的蛋白質降解、使SSR內成為高度蛋白質聚集的區域，當神經元活性降低時，後突觸細胞內的cAMP/PKA活性下降，使得蛋白酶體與Ecm29的交互作用受到抑制，造成蛋白酶體解體、GluRIIA在突觸處快速累積，因而產生較大的後突觸反應，補償降低的神經元活性。由此假說推測可能在後突觸附近有未知的運輸系統可辨認並將GluRIIA等需經由蛋白酶體降解的物質由SSR內送出，此假設尚需要更多的實驗來證明。

In response to environmental and physiological changes, the synapses manifest plasticity while simultaneously maintain homeostasis. A well-tuned proteome is required to meet these demands which is maintained with sufficient de novo protein synthesis and transport while mis-folded and impaired proteins are removed. In the first part, we analyzed mutant synapses of henji, also known as dbo, at the Drosophila neuromuscular junctions (NMJs). In henji mutants, NMJ growth is defective with appearance of satellite boutons. Transmission electron microscopy analysis indicates that the synaptic membrane region is expanded. The postsynaptic density (PSD) houses glutamate receptors GluRIIA and GluRIIB, which have distinct channel transmission properties. In henji mutants, GluRIIA abundance is upregulated but that of GluRIIB is not. Electrophysiological results also support a GluR compositional shift towards a higher IIA/IIB ratio at henji NMJs. Strikingly, dPAK, a positive regulator for GluRIIA synaptic localization, accumulates at henji PSDs. Reducing the dpak gene dosage suppresses satellite boutons and GluRIIA accumulation at henji NMJs. In addition, dPAK associated with Henji through the Kelch repeats which is the domain essential for Henji localization and function at postsynapses. We propose that Henji acts at postsynapses to restrict both presynaptic bouton growth and postsynaptic GluRIIA abundance by modulating dPAK. In the second part, we found that the proteasome is absent in the subsynaptic reticulum (SSR) of Drosophila NMJs where synaptic proteins are enriched. This specific “cold zone” is not shaped by structural barrier of the SSR, instead, proteasome is anchored by the adaptor protein Extracellular mutant 29 (Ecm29), whose localization is excluded from the SSR. When the proteasome activity is transiently inhibited by RNAi for essential subunits of the 19S regulatory particle, GluRIIA accumulates while GluRIIB does not. Rapid modulation on GluRIIA synaptic levels enables the postsynapses to respond to changes in synaptic activity. Indeed, when neuronal activity is transiently inhibited, synaptic proteins sensitive to proteasome activity readily accumulated. Activating the cAMP/PKA pathway suppresses this activity withdrawal-triggered GluRIIA accumulation. From above data, we propose a model that the proteasomes are segregated from the SSR by anchoring to Ecm29 to preserve high local concentration of synaptic proteins inside the SSR. When neuronal activity is drastically reduced, the postsynapse responds by reducing the proteasome activity via inhibiting the cAMP/PKA pathway and thus promotes GluRIIA synaptic levels to compensate the drop in activity. An unidentified pathway may be involved in transferring GluRIIA and other proteasome substrates out of the SSR to the proteasome active region for degradation.