臺灣博碩士論文加值系統

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。

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.

Table of contents
口試委員會審定書 i
摘要 ii
Abstract iii
Chapter 1 Introduction 1
1.1 Drosophila locomotor circuit 1
1.2 Excitatory Amino Acid Transporter (EAAT) and the neurological disorders 1
Chapter 2 Material and methods 4
2.1 Drosophila strains 4
2.2 Identification of molecular lesion in the EMS-mutagenized mutants 7
2.3 Generation of Clc-c null mutant via P-element excision 11
2.4 Generation of transgenic fly 11
2.5 Immunohistochemistry 12
2.6 Western blotting 14
2.7 Fly genetics 15
2.8 Extracellular glutamate measurement in the VNC by glutamate sensor iGluSnFR 16
2.9 Electrophysiology 17
2.10 Calcium imaging 17
2.11 Video tracking of larval locomotion 18
2.12 Drug treatment 18
2.13 Mitochondrial stress measurement by MitoTimer 19
2.14 In vivo detection of reactive oxygen species by H2DCFDA 19
2.15 Statistical analysis 20
Chapter 3 Results 3
3.1 A forward genetic screen for novel players involved in the formation of synaptic boutons at the Drosophila NMJs 21
3.2 The mutation in eaat1 promotes the formation of the NMJ boutons 23
3.3 Expression of Eaat1 in astrocytes is responsible for the formation of NMJ boutons 24
3.4 Eaat1-mediated glutamate uptake in astrocytes is responsible for rhythmicity of CPG activity and locomotion 25
3.5 Glutamate accumulation at the synaptic clefts of excitatory and inhibitory glutamatergic interneurons neurons impair CPG activity 28
3.6 Loss of eaat1 results in oxidative stress in cholinergic interneurons, which impedes the rhythmicity of CPG 31
3.7 Impaired CPG activity non-autonomously alters the formation of the NMJ boutons in the eaat1 mutants 33
3.8 ROS-mediated JNK activation autonomously promotes the formation of the NMJ boutons in the eaat1 mutants 34
Chapter 4 Discussion 36
4.1 Conserved role of Drosophila Eaat1 on glutamate transmission and locomotor behavior 36
4.2 CPG firing pattern and the synaptic bouton formation in the NMJ 37
4.4 Aberrant neural circuit activity in neurological disorder 38
Figures 39
Figure 1. Cases of different types of neurons involved in Drosophila larval locomotor circuit 39
Figure 2. Representation of Drosophila larval neuromuscular junction (NMJ) 41
Figure 3. Characterization and deficiency mapping for the molecular lesion of the EMS-mutagenized mutant collection 42
Figure 4. BE471 is a strong hypomorphic eaat1 allele and responsible for the excessive synaptic boutons formation at Drosophila larval NMJ 44
Figure 5. Quantification of NMJ phenotype associated with eaat1BE/BE at muscle 4 46
Figure 6. Eaat1 is severely reduced in the CNS of 3rd instar eaat1BE/BE mutant larva 47
Figure 7. Synaptic bouton size and density of active zone are altered in eaat1BE/BE mutant 48
Figure 8. Expression profile of Eaat1 in 3rd instar larval VNC 50
Figure 9. Eaat1 is not expressed in Drosophila 3rd instar larval NMJ 51
Figure 10. Astrocyte-associated Eaat1 is responsible for excessive synaptic boutons formation at NMJ associated with eaat1BE/BE 52
Figure 11. Astrocyte-associated Eaat1 regulates the activity of CPG 54
Figure 12. Astrocyte-associated Eaat1 controls the larval locomotor activity 56
Figure 13. Eaat1 activity regulates the larval peristaltic locomotion 57
Figure 14. Eaat1 is wildly distributed in the processes of astrocyte-like glia which are closely associated with glutamatergic presynapses and responsible for extracellular glutamate clearance. 58
Figure 15. Efficacy of vGlut-RNAi on extracellular glutamate level. 60
Figure 16. Excessive extracellular glutamate in the CNS is responsible for aberrant CPG activity and locomotor deficits associated with eaat1BE/BE mutant. 62
Figure 17. Inhibitory glutamatergic neurons partly contribute to the locomotor deficits in eaat1BE/BE mutants. 65
Figure 18. Oxidative stress is mildly elevated in eaat1BE/BE mutant 66
Figure 19. Elevated oxidative stress in central cholinergic neurons contribute to aberrant CPG activity and locomotor deficits in eaat1BE/BE mutant 68
Figure 20. The abnormal CPG activity and locomotor deficits associated with eaat1BE/BE can be partly rescued by increasing the excitability in central cholinergic neurons. 71
Figure 21. The excessive synaptic boutons formation at NMJ in eaat1BE/BE was associate with aberrant CPG activity. 73
Figure 22. Mitochondria in eaat1BE/BE is suffered from elevated oxidative stress 75
Figure 23. ROS-induced JNK signaling in motoneuron is responsible for excessive synaptic boutons formation at NMJ in eaat1BE/BE mutant 76
Figure 24. The pattern of CPG activity is dependent on Eaat1 level 78
Figure 25. BMP signal is selectively activated in eaat1 mutant with severe loss of eaat1 80
Figure 26. Schematic model of glutamate excitotoxicity on Drosophila larval motor circuit in loss of eaat1 82
Tables 84
Table 1. Summary of the EMS mutants whose corresponding gene for NMJ phenotype were identified 84
Table 2. Summary of the forward genetic screen for novel players involved in synaptic formation at Drosophila larval NMJ 85
Contribution 87
Reference 88

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