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

Rab27 regulates neurotransmission through Unc13 in mushroom body α/β posterior neurons for lifespan extension in Drosophila

李易展(Yi-Jhan Li)

指導教授 ： 詹智強

國立臺灣大學/醫學院/生理學研究所/碩士(2020年)

https://doi.org/10.6342/NTU202000432

摘要

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。

關鍵字

果蠅 ； 壽命延長 ； 蕈狀體 ； Rab27 ； Unc13 ； 神經傳遞

並列摘要

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.

並列關鍵字

Drosophila ； lifespan extension ； mushroom body ； Rab27 ； Unc13 ； neurotransmission

參考文獻

1. Alcedo, J. and C. Kenyon, Regulation of C-elegans longevity by specific gustatory and olfactory neurons. Neuron, 2004. 41(1): p. 45-55.

2. Libert, S., et al., Regulation of Drosophila life span by olfaction and food-derived odors. Science, 2007. 315(5815): p. 1133-1137.

3. Waterson, M.J., et al., Water sensor ppk28 modulates Drosophila lifespan and physiology through AKH signaling. Proceedings of the National Academy of Sciences of the United States of America, 2014. 111(22): p. 8137-8142.

4. Riera, C.E., et al., TRPV1 Pain Receptors Regulate Longevity and Metabolism by Neuropeptide Signaling. Cell, 2014. 157(5): p. 1023-1036.

5. Satoh, A., et al., Sirt1 Extends Life Span and Delays Aging in Mice through the Regulation of Nk2 Homeobox 1 in the DMH and LH. Cell Metabolism, 2013. 18(3): p. 416-430.