Rab27於神經系統調控壽命之機制

Abstract

大腦是老化調節中樞，壽命長短會受大腦調控，但卻少有研究討論特定腦區內調控壽命的分子機制及其可能的神經迴路。本論文以果蠅為模式動物，著重於腦神經調控壽命的機制及迴路。果蠅大腦中的蕈狀體相當於哺乳類的海馬迴以及下視丘。實驗結果顯示，在果蠅蕈狀體抑制rab27會促進長壽。因此我們在特定腦區進行核糖核酸干擾作用，建立Rab27在蕈狀體調控壽命的神經迴路，從大腦單側2000顆神經元構成的蕈狀體，縮小範圍剖析至大腦單側約73顆α/β posterier神經元。在蔗糖濃度梯度分層實驗中發現，rab27KO造成磷酸化態核糖體蛋白S6激酶（p-S6K）的分層位置明顯位移，進一步利用免疫沉澱法及蛋白質交互作用鄰位連接法證明Rab27與p-S6K具有直接交互作用。再從免疫螢光染色結果得知，無論在α/β posterier神經元中的軸突或樹突皆觀察到p-S6K蛋白量明顯減少。此外，只在α/β posterier神經元過表達非活化態s6k (s6kDN) 就可有效延長壽命。我們也發現α/β posterier神經活性會隨著年紀明顯增加，只要操弄α/β posterier神經特性就會明顯改變壽命。有趣的是，rab27KO會降低α/β posterier神經活性，以及影響α/β posterier神經元內蛋白新生能力。更重要的是，只在α/β posterier神經元中過表達仿磷酸化S6 (phosphor-mimetic S6) 會顯著回復rab27KO造成的壽命延長及蛋白新生成降低。因此，我們推論Rab27調控α/β posterier神經活性，並經由TOR訊息路徑影響蛋白新生成，進而維持正常的壽命。另外，我們利用GS果蠅株過表達目標基因進行壽命調節基因篩選，從果蠅資料庫挑選會對應到人類同源基因的GS影響基因，再藉由rab27KO延長壽命的優勢，在rab27KO背景中，只在rab27表現細胞中過表達GS影響基因，篩選出顯著影響壽命的基因。實驗發現，在這些顯著影響壽命的基因中，我們篩選到數個已知調控壽命的基因，以及不同GS株卻影響到相同基因，說明我們建立的果蠅壽命基因篩選系統，可公平篩選出每一個影響壽命的人類同源基因。本研究對於探討未知的人類同源基因在中樞神經系統中如何相互作用維持個體壽命，可起開端之效。

The nervous system is a major tissue to modulate lifespan. The mammalian CNS has been proposed to regulate aging; however, the minimal sufficient region and the underlying mechanism for neuronal regulation of longevity remain unknow. In the first part, we show that the Drosophila lifespan is modulated by rab27, encoding an evolutionarily conserved Rab GTPase mediating the exocytic machinery, and functioning in a small subset of neurons of the mushroom bodies (MB), a brain structure which shares analogous functions with mammalian hippocampus and hypothalamus. Depleting rab27 in the α/β posterior neurons of the MB is sufficient to extend lifespan, enhance systemic stress responses, and alter energy homeostasis, all without trade-offs in major life functions. Within the α/β posterior neurons, rab27KO causes the mislocalization of phosphorylated S6K thus attenuates TOR signaling, resulting in decreased protein synthesis and reduced neuronal activity. Consistently, expression of dominant-negative S6K in the α/β posterior neurons increases lifespan. Furthermore, expression of phospho-mimetic S6 in α/β posterior neurons of rab27KO rescued local protein synthesis and reversed lifespan extension. These findings demonstrate that inhibiting TOR-mediated protein synthesis in α/β posterior neurons is sufficient to promote longevity. In the second part, we performed a modifier screen to identify novel Drosophila longevity genes with human homologs. This screen identified potential enhancers and suppressors whose overexpression in rab27KO background regulate longevity. Some genes have been previously reported to regulate lifespan, validating that this screening approach for candidate genes is feasible. Our findings demonstrate that genes regulating functions of rab27-expressing neurons may be critical in human longevity traits, and await further investigation.