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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 詹智強 | |
dc.contributor.author | Yi-Jhan Li | en |
dc.contributor.author | 李易展 | zh_TW |
dc.date.accessioned | 2021-06-17T00:23:10Z | - |
dc.date.available | 2020-03-12 | |
dc.date.copyright | 2020-03-12 | |
dc.date.issued | 2020 | |
dc.date.submitted | 2020-02-11 | |
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Barnstedt, O., et al., Memory-Relevant Mushroom Body Output Synapses Are Cholinergic. Neuron, 2016. 89(6): p. 1237-1247. 26. Jing, M., et al., A genetically encoded fluorescent acetylcholine indicator for in vitro and in vivo studies. Nature Biotechnology, 2018. 36(8): p. 726-+. 27. Sun, F.M., et al., A Genetically Encoded Fluorescent Sensor Enables Rapid and Specific Detection of Dopamine in Flies, Fish, and Mice. Cell, 2018. 174(2): p. 481-+. 28. Marvin, J.S., et al., An optimized fluorescent probe for visualizing glutamate neurotransmission. Nature Methods, 2013. 10(2): p. 162-170. 29. Aso, Y., et al., The neuronal architecture of the mushroom body provides a logic for associative learning. Elife, 2014. 3. 30. Chan, C.C., et al., Systematic Discovery of Rab GTPases with Synaptic Functions in Drosophila. Current Biology, 2011. 21(20): p. 1704-1715. 31. Bruinsma, S., et al., Small molecules that inhibit the late stage of Munc13-4-dependent secretory granule exocytosis in mast cells. 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Kanno, Analysis of the role of Rab27 effector Slp4-a/granuphilin-a in dense-core vesicle exocytosis, in Gtpases Regulating Membrane Targeting and Fusion, W.E. Balch, C.J. Der, and A. Hall, Editors. 2005. p. 445-457. 43. Fukuda, M., Slp4-a/granuphilin-a inhibits dense-core vesicle exocytosis through interaction with the GDP-bound form of Rab27A in PC12 cells. Journal of Biological Chemistry, 2003. 278(17): p. 15390-15396. 44. Chiba, T., et al., A key role for neuropeptide Y in lifespan extension and cancer suppression via dietary restriction. Scientific Reports, 2014. 4. 45. Ostenfeld, M.S., et al., Cellular Disposal of miR23b by RAB27-Dependent Exosome Release Is Linked to Acquisition of Metastatic Properties. Cancer Research, 2014. 74(20): p. 5758-5771. 46. Tang, L., D. Wei, and F. Yan, MicroRNA-145 functions as a tumor suppressor by targeting matrix metalloproteinase 11 and Rab GTPase family 27a in triple-negative breast cancer. Cancer Gene Therapy, 2016. 23(8): p. 258-265. 47. Grosshans, B.L., D. Ortiz, and P. Novick, Rabs and their effectors: Achieving specificity in membrane traffic. Proceedings of the National Academy of Sciences of the United States of America, 2006. 103(32): p. 11821-11827. 48. Ostermeier, C. and A.T. Brunger, Structural basis of Rab effector specificity: Crystal structure of the small G protein Rab3A complexed with the effector domain of Rabphilin-3A. Cell, 1999. 96(3): p. 363-374. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/66138 | - |
dc.description.abstract | 生物體的老化經常伴隨著神經系統的退化,而神經系統也在調節壽命方面扮演重要的角色。從實驗室的研究得知,在果蠅蕈狀體αβ posterior(αβp)神經群降低rab27的表現量,會造成壽命延長與有較強的抗飢餓能力;另外在rab27KO情況下,會造成原先與Rab27有交互作用的p-S6K錯位,進一步使的磷酸化S6減少,因此TOR訊息活性被抑制,而造成壽命延長。
Rab27是Rab GTPase的一員,當其活化時會以GTP結合的形式,並在不同的情境下與對應的效應子蛋白相互作用,舉例來說,文獻指出在哺乳類的T细胞、血小板與嗜中性白血球,Munc13皆會與GTP-Rab27共同調控胞吐作用,而在果蠅中,Unc13雖與Munc13在胺基酸序列上有67%的相似性,但是否為Rab27的效應子蛋白,且能在αβp神經群調控壽命,並無文獻指出。在我的實驗中,首先藉由Co-IP測試在果蠅腦神經中,Unc13與Rab27具有交互作用,且在Rab27為持續活化態時,Unc13會與Rab27有較強的交互作用;另外利用PLA assay,發現在αβ posterior區域,Unc13與Rab27同樣有蛋白質交互作用,而在果蠅蕈狀體αβp神經群降低unc13的表現量,也會造成壽命的延長,並有較強的抗飢餓能力。 Rab27主要促進胞吐作用,且在果蠅體內高度表現於神經突觸囊泡,先前文獻也指出果蠅體內神經突觸間的傳遞,仰賴Unc13對囊泡的調控,因此我們推測Rab27在果蠅的αβp神經群裡,可能調控了某些物質於突觸間的分布,例如神經傳遞物質、神經肽、激素或是mircoRNA等等,因此影響果蠅壽命。 首先,我們發現在失去rab27的情況下,會造成老化果蠅αβp區域神經細胞內部與外部的GABA量增加,年輕果蠅則無,顯示出在αβp神經群確實有神經傳遞物質會受Rab27的調控,且為一漸進式的現象。除了GABA的免疫染色實驗之外,我的實驗中還使用了UAS-neurotransmitter sensor-GFP果蠅,藉由這些神經傳遞物質sensor來測試在失去rab27時,哪些神經傳遞物質的多寡會在αβp神經群的軸突突觸受到影響。我們發現在rab27KO的情況下,在αβp神經群,無論年輕或老化的果蠅,多巴胺與乙醯膽鹼的GFP亮度皆是增加的,麩胺酸則沒有改變。 根據上述結果,同樣利用GABA免疫染色實驗與神經傳遞物質sensor,來測試在αβ lobe的下游神經共6種output neurons (MBONs) 的樹突處,哪些神經傳遞物質的多寡,可能因為失去rab27而受到影響。我們發現在rab27KO的情況下,於MBON-β1>α樹突處的GABA量,無論年輕或老化的果蠅皆是下降的,而在MBON-γ1pedc>α/β樹突處,多巴胺的GFP亮度於年輕時為增加,老化時則減少。綜合上述結果,顯示出在αβp神經群與αβ lobe的下游神經區域確實有神經傳遞物質會因失去rab27而受到影響,且可能有不同面向。 | zh_TW |
dc.description.abstract | Aging is the progressive degeneration of the nervous system, which also plays an important role in modulating longevity. Recent work from our lab has revealed that knockdown of rab27 in αβ posterior (αβp) neurons extends longevity. Moreover, rab27KO caused p-S6K mislocalization and decreased the level of p-S6, resulting in the deactivation of Tor and lifespan extension.
Rab27 is a member of the Rab subfamily of GTPases, which is activated when bound to GTP, and associated with corresponding effector in different cell types. For example, studies have shown that mammalian Unc13 (Munc13) is a GTP-Rab27-binding protein, which regulates vesicle trafficking in T cells, platelets and neutrophil granules. In Drosophila, Unc13 has a 67% similarity to Munc13 in their amino acid sequence, however, research have not shown that Unc13 is Rab27 effector in Drosophila, and whether it regulates lifespan in the αβp neurons is unknown. In my experiments, I found that Unc13 co-immunoprecipitated with Rab27 and is more associated with the constitutively activated form of Rab27. Also, I found a direct association between Rab27 and Unc13 in the αβp neurons by PLA assay. Moreover, knockdown of unc13 in αβp neurons also extends flies longevity and induced them to be highly resistant to starvation stress. Rab27 plays a key role in regulating exocytosis, and is highly expressed in synaptic vesicles in Drosophila. In addition, Drosophila Unc-13 is essential for synaptic transmission, therefore, we speculated that Rab27 may regulate the level of certain substances such as neurotransmitters, neuropeptides, hormones, or mircoRNAs in the αβp neurons synapse, thus affecting the lifespan. First, we found that the intensity of GABA level is increased in both inside and outside of αβp neurons axon in aged rab27KO, but not in young flies, indicating that GABA transmission is regulated by Rab27 in αβp neurons and this regulation occurs in a progressive phenomenon. In addition to GABA immunostaining, I also used UAS-neurotransmitter sensor-GFP in my experiments to test which neurotransmitters are affected in axon synapses of rab27KO αβp neurons. I found that in rab27KO, the GFP intensity of dopamine and acetylcholine sensors both increased in young and old flies, while no affected in glutamate sensor. According to the results above, I also used GABA immunostaining and neurotransmitter sensor-GFP to test the dendrites of 6 output neurons (MBONs) downstream of αβ lobe, and investigated potential neurotransmitters that may be affected in rab27KO. I found that in rab27KO, the intensity of GABA level is decreased in MBON-β1>α dendrites regardless of young or aged flies. While in MBON-γ1pedc>α/β dendrites, intensity of dopamine GFP is increased in young but decreased in aged flies, indicating that the neurotransmitters in the downstream of αβ lobe might be also regulated by Rab27, and have different orientations. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T00:23:10Z (GMT). No. of bitstreams: 1 ntu-109-R06441016-1.pdf: 6793358 bytes, checksum: 9ed6587bb909c75429ece48d061bea95 (MD5) Previous issue date: 2020 | en |
dc.description.tableofcontents | 致謝 ii
中文摘要 iii 英文摘要 v 目錄 vii 第一章 實驗背景 1 1.1 神經系統對壽命的調控 2 1.2 果蠅蕈狀體神經元對於壽命的調控 3 1.3 Rab27與效應子蛋白(effector)在生物體中的功能 4 1.4 神經傳遞物質與壽命延長之訊息傳遞路徑的關係 5 1.5 蕈狀體神經迴路 6 1.6 Hypothesis 7 1.7 Specific Aims 7 第二章 實驗材料與方法 9 2.1 主要抗體與果蠅株表格 10 2.2果蠅飼養方法 11 2.3存活取線分析 11 2.4免疫螢光染色 11 2.5 鄰位連接技術(PLA) 13 2.6 Quickchange PCR 14 2.7 免疫共沉澱(Co-IP) 15 2.8 Neurotransmitter Sensors 17 2.9 TAG、glucose測量 17 2.10 Bexin-1 餵食 18 第三章 實驗結果 19 3.1 Unc13與Rab27在αβp神經群中具有蛋白交互作用 20 3.2 Unc13在腦神經中具Rab27效應子蛋白之特性 20 3.3 在αβp神經群減少unc13的表現量造成果蠅壽命延長並有較強之抗挨餓能力 20 3.4 在αβp神經群減少unc13的表現量並無觀察到果蠅代謝上的改變 21 3.5 餵食果蠅Munc13 inhibitor- Bexin1並無觀察到果蠅壽命延長 21 3.6 失去rab27造成αβp神經群軸突突觸,GABA、多巴胺與乙醯膽鹼含量增加 22 3.7 在αβp神經群降低Gad1表現量不影響年輕或老化果蠅於壓力環境下壽命 23 3.8 失去rab27造成αβ lobe輸出神經群樹突處,GABA、多巴胺與麩胺酸含量改變 23 第四章 實驗討論 25 4.0 Summary of my findings 26 4.1 Significance 26 4.2 Rab27與Rab3互相調控胞吐作用 27 4.3 果蠅蕈狀體的神經傳遞物質輸出與神經傳遞物質受器 27 4.4 Rab27可能運輸之物質(神經肽、micro RNA等) 28 4.5 MB – lifespan circuit 29 第五章 未來實驗方向 30 5.1 Rab – effector biochemical interaction 31 5.2 Rab27KO是否於αβp神經群藉由神經傳遞物質受器調控壽命 31 5.3 UAS-neurotransmitter-sensor GFP的活體測試 31 第六章 實驗圖表 33 Fig. 1 Rab27與Unc13在果蠅蕈狀體αβ posterior神經群具有交互作用 34 Fig. 2 Rab27持續活化態 (Rab27-CA) 相較於Rab27-WT與Unc13在腦部神經有較高比例交互作用 35 Fig. 3 在蕈狀體αβ posterior神經群降低unc13表現量造成壽命延長 36 Fig. 4 在蕈狀體αβ posterior神經群降低unc13表現量造成老化果蠅於壓力環境下有較長壽命 37 Fig. 5 在蕈狀體αβ posterior神經群降低unc13,能造成αβ posterior神經群軸突處Uc13蛋白量減少 38 Fig. 6 在蕈狀體αβ posterior神經群降低unc13,並無觀察到果蠅代謝上的改變 39 Fig. 7 餵食果蠅Munc13 inhibitor- Bexin1並無觀察到果蠅壽命延長 40 Fig. 8 年輕rab27KO果蠅,在αβ posterior神經群中GABA含量沒有差異;老化rab27KO果蠅,則在αβ posterior神經群中有較高GABA含量 41 Fig. 9 年輕與老化rab27KO果蠅,在αβ posterior神經群軸突突觸,多巴胺sensor-GFP亮度皆有增加趨勢 42 Fig. 10 年輕與老化rab27KO果蠅,在αβ posterior神經群軸突突觸,乙醯膽鹼sensor-GFP亮度有增加趨勢 43 Fig. 11 年輕與老化rab27KO果蠅,在αβ posterior神經群軸突突觸,麩胺酸sensor-GFP亮度不受影響 44 Fig. 12 在蕈狀體αβ posterior神經群降低Gad1表現量,不影響年輕或老化果蠅於壓力環境下壽命 45 Fig. 13 MBON Gal4 lines表現位置 46 Fig. 14 年輕與老化之rab27KO果蠅,在MBON-α3與MBON-α1神經群樹突突觸,多巴胺sensor-GFP亮度沒有差異 47 Fig. 15 年輕rab27KO果蠅,在MBON-γ1pedc>α/β神經群樹突突觸,多巴胺sensor-GFP亮度有增加趨勢; 老化rab27KO果蠅,在MBON-γ1pedc>α/β神經群樹突突觸,多巴胺sensor-GFP亮度則有減少趨勢 49 Fig. 16 年輕與老化果蠅,在MBON神經群樹突突觸,無法清楚偵測到乙醯膽鹼sensor-GFP亮度 50 Fig. 17 年輕與老化rab27KO果蠅,在MBON-α3與MBON-γ1pedc>α/β神經群樹突突觸,麩胺酸sensor-GFP亮度沒有差異 51 Fig. 18 年輕與老化rab27KO果蠅,在MBON-α1神經群樹突突觸,麩胺酸sensor-GFP亮度皆有減少趨勢 53 Fig. 19 年輕與老化rab27KO果蠅,在MBON-α3與MBON-α1神經群樹突處,GABA含量沒有差異 54 Fig. 20 年輕與老化rab27KO果蠅,在MBON-β1>α神經群樹突處,GABA含量皆有減少趨勢,MBON-β2則無影響 56 Fig. 21 利用鄰位連接技術(PLA)偵測蛋白蛋白交互作用示意圖 58 Fig. 22 神經傳遞物質sensor-GFP應用示意圖 59 Table. 1 年輕與老化之rab27KO,於αβp神經群與MBON突觸,調控之神經傳遞物質 60 Table. 2 UAS-S6-S2A-19 定序結果 61 Table. 3 UAS-S6-S2A-12 定序結果 61 Table. 4 UAS-S6-S2D-20 定序結果 62 Table. 5 UAS-S6-S2D-32 定序結果 62 Table. 6 UAS-S6-S5A-04 定序結果 63 Table. 7 UAS-S6-S5A-10 定序結果 63 Table. 8 UAS-S6-S5D-01 定序結果 64 Table. 9 UAS-S6-S5D-06 定序結果 64 Table. 10 UAS-S6-WT-02 定序結果 65 Table. 11 UAS-S6-WT-23 定序結果 65 第七章 參考文獻 66 | |
dc.language.iso | zh-TW | |
dc.title | 探討Rab27與Unc13在果蠅蕈狀體中,藉由神經傳遞物質調控壽命的可能機制 | zh_TW |
dc.title | Rab27 regulates neurotransmission through Unc13 in mushroom body α/β posterior neurons for lifespan extension in Drosophila | en |
dc.type | Thesis | |
dc.date.schoolyear | 108-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 汪宏達,吳嘉霖,林子暘 | |
dc.subject.keyword | 果蠅,壽命延長,蕈狀體,Rab27,Unc13,神經傳遞, | zh_TW |
dc.subject.keyword | Drosophila,lifespan extension,mushroom body,Rab27,Unc13,neurotransmission, | en |
dc.relation.page | 70 | |
dc.identifier.doi | 10.6342/NTU202000432 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2020-02-12 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 生理學研究所 | zh_TW |
顯示於系所單位: | 生理學科所 |
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