探討果蠅Eaat1透過清除穀胺酸在整合運動迴路運轉及神經肌肉接合點形成的角色

Investigate roles of Drosophila Eaat1-mediated glutamate clearance in the functional integrity of the motor circuit and the formation of the neuromuscular junction

彭湛傑(Jhan-Jie Peng)

指導教授 ： 姚季光

國立臺灣大學/生命科學院/生化科學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201602813

摘要

在中樞神經系統中，穀胺酸訊息傳遞對於動物行為的調控是不可或缺的。一旦穀胺酸從突觸囊泡中釋放，它需要立刻被周圍的星狀膠細胞給移除。星狀膠細胞上的刺激性胺基酸載體(Eaat)負責回收大部分的穀胺酸，藉此確保穀胺酸訊息傳遞的精確性以及避免穀胺酸過度刺激所產生的毒性。在神經退化性疾病致中，常伴隨著Eaat1表現量的下降，而回收機制的失衡將導致穀胺酸累積在細胞外，神經系統因此被過度刺激而加速疾病的惡化。雖然穀胺酸如何導致神經細胞死亡的機制已被廣泛的研究，但它的刺激毒性對於神經迴路整體的影響卻不清楚。因此，本篇研究利用果蠅幼蟲的運動迴路系統，探討星狀膠細胞在穀胺酸訊息傳遞的角色。從找尋參與突觸生成的新分子的遺傳篩選實驗中，我發現了果蠅Eaat1的減效對偶基因。在果蠅幼蟲中，此突變的對偶基因導致Eaat1的表現大幅下降，神經肌肉接合點的突觸終釦大量增生，幼蟲的運動能力也大受影響。果蠅幼蟲的蠕動爬行是受到中樞樣式產生器(CPG)的神經迴路所調控，Eaat1的喪失導致異常的CPG活性，受其調控的運動神經元雖然變得不常活化，但每次活化的時間卻明顯拉長。在eaat1突變果蠅中，單獨將Eaat1表現在星狀膠細胞中, 便能救回異常的CPG活性以及爬行能力。Eaat1的喪失也伴隨著細胞外穀胺酸的累積，干擾了刺激性及抑制性神經在CPG中的訊息傳遞，因而影響幼蟲的爬行能力。值得注意的是，降地刺激性乙醯膽鹼神經的氧化壓力或著增加它的刺激性能夠顯著改善CPG的活性以及幼蟲的爬行能力。此外，異常的CPG活性造成下游的運動神經產生大量的過氧化物，受環境壓力所調控的JNK訊息路徑因此被活化，導致突觸終釦的異常增生。因此，本篇研究闡述位於運動迴路上游的星狀膠細胞，如何藉由Eaat1對於迴路活性的影響，來調控運動神經元的特性。

關鍵字

穀胺酸訊息傳遞 ； 刺激毒性 ； 星狀膠細胞 ； 刺激性胺基酸載體1 ； 中樞樣式產生器 ； ROS/JNK訊息路徑 ； 神經肌肉接合點

並列摘要

Glutamate transmission in CNS is critical for animal behavior. Upon the release of glutamate from synaptic vesicles, it is immediately removed by the surrounding astrocyte. The Excitatory Amino Acid Transporter (Eaat) in astrocyte is majorly responsible for the glutamate clearance in CNS, which ensures the fidelity of neurotransmission and prevents glutamate excitotoxicity. In neurodegenerative diseases, downregulation of Eaat has been repeatedly reported and accumulation of extracellular glutamate due to impaired recycle system accelerates the disease progression. Although how excessive glutamate leads to neuronal death has been widely studied, the influence of glutamate excitotoxicity on neural circuit is still elusive. Here, we use Drosophila larval locomotor circuit to study the role of glia on the glutamate transmission in CNS. From a genetic screen for novel players involved in synaptic formation, we identified a strong hypomorphic Drosophila eaat1 allele which led to strong loss of eaat1, expanded neuromuscular junction, and severe locomotor deficits. The locomotion of Drosophila larva is controlled by the neural circuit called central pattern generator (CPG). Loss of eaat1 resulted in aberrant CPG activity which caused prolonged but less frequent activation of motoneurons. Restoring the expression of eaat1 exclusively in the astrocyte was sufficient to restore the CPG and locomotion defects in eaat1 mutants. Impaired glutamate clearance in loss of eaat1 led to extracellular glutamate accumulation and disrupted the excitatory and inhibitory synaptic signaling in the CPG. Remarkably, reducing the oxidative stress or increasing the excitability of excitatory cholinergic neurons in eaat1 mutants improved both CPG rhythmicity and mobility. Moreover, the irregular CPG firing pattern in loss of eaat1 triggered the ROS/JNK signaling in motorneurons and caused excessive synaptic bouton formation. We demonstrated a non-cell autonomous mechanism to regulate the physiology of motoneurons via Eaat1 in upstream astrocyte.

並列關鍵字

Glutamate transmission ； Excitotoxicity ； Astrocyte ； Eaat1 ； CPG ； ROS/JNK signaling ； NMJ

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