運用細胞專一性基因表現技術探討催產素由下視丘神經垂體系統釋放的分子機制

Abstract

催產素(OT)由下視丘-神經垂體系統中的室旁核(PVN)及視上核(SON)的magnocellular neurons (MCNs)所製造。最初，催產素被發現具有調控生理方面的功能；隨著對催產素有更深入的研究，發現催產素還具有調控情緒反應以及社交行為等功能。然而，調控如此重要神經胜肽的釋放機制卻仍然不明確。目前已知，位於催產素神經元軸突末梢的腦下垂體後葉有兩種類型的囊泡，一種為大小約100-300奈米，具有緻密核心的large dense-core vesicles (LDCVs)，另一種為型態及生化特性都與synaptic vesicles (SVs)相似的microvesicles (MVs)。這些囊泡除了能夠受到Ca2+-dependent exocytosis所調控。SV/MV上的Syn Ia也能夠藉由磷酸化與否來調控囊泡的釋放。因此，我們想探討專一表現在SV/MV上的Syn Ia除了能夠調控SV/MV的釋放，是否也能夠調控LDCV的釋放，進而使催產素的釋放量上升。因此，我們首先設計了一系列由催產素啟動子所驅動的囊泡報導蛋白(VAMP2-pHVenus or neurophysin I-pHVenus)並帶有Syn Ia或是Syn Ia突變體(S62A)的DNA。接著，我們利用活體電穿孔(in vivo electroporation)的方式將這些DNA轉染進入成年雄性大鼠的SON中。為了觀察DNA在SON當中表現的情形，我們使用免疫螢光染色來標定在SON中表現的囊泡報導蛋白。我們發現兩種囊泡報導蛋白都能夠成功表現，而neurophysin I-pHVenus所表現的囊泡型態較VAMP2-pHVenus更大，並且訊號和催產素更加重疊。除了螢光染色外，我們也利用酵素連結免疫吸附測定(ELISA)來檢測實驗前後催產素在腦脊髓液(CSF)及血清(plasma)中的濃度。我們發現Syn Ia能夠提高血清中的催產素濃度，而在腦脊髓液中則沒有太大改變。除此之外，我們也檢測了與催產素結構相似的血管加壓素(VP)在血清中的濃度變化。有趣的是，血管加壓素在實驗後的濃度也提高了；並且使得大鼠保留了更多水分在體內而使得體重有些微的上升，我們認為這或許是有部分的DNA被轉染進入SON中一種能夠同時表現催產素及血管加壓素的神經元中所造成。總結我們的實驗結果顯示，催產素專一性的DNA能夠專一的被表現在SON中的催產素神經元，且Syn Ia能夠使釋放到血清中的催產素濃度上升。

Oxytocin (OT), synthesized from the magnocellular neurons (MCNs) of paraventricular nucleus (PVN) and supraoptic nucleus (SON) in hypothalamic-neurohypophysis system (HNS), is initially found to regulate physiological functions in mammalian species. Along with the deep investigation of OT, this nonapeptide can also regulate emotional responses and social behaviors. However, the mechanism regulating the OT release remains unclear. The axon terminals of OT neurons in posterior pituitary contain two types of vesicles. One is called large dense-core vesicles (LDCVs), 100-300 nm in diameter, containing dense particles. The other is called microvesicles (MVs) which are morphologically and biochemically similar to synaptic vesicles (SVs). Though the exocytosis of both vesicles are Ca2+-dependent, a SV/MV-specific protein Syn Ia is only expressed on synaptic vesicles to regulate the exocytosis in a phosphorylation-dependent manner. Here, we aimed to determine the role of Syn Ia in OT neurons and examine whether the SV/MV-specific protein can also regulate the exocytosis of LDCVs. We first designed a series of OT neuron-specific constructs containing the exocytosis reporter and Syn Ia/its mutant. We next expressed the constructs in OT neurons of SON by using in vivo electroporation. To detect the expression of the constructs, we performed immunostaining and labeled the exocytosis reporter by the antibody against green fluorescent protein (GFP). In addition, the patterns of immunoreactivity in the LDCV-specific reporter were larger than the general vesicle reporter, with relatively localized to OT. Furthermore, we also detected the OT levels in CSF and plasma before and after in vivo electroporation by using ELISA. We found that the overexpression of Syn Ia can increase the release of OT to plasma rather than to CSF. However, the overexpression of Syn Ia mutant cannot significantly affect the release of OT to either plasma or CSF. In addition to detecting the levels of OT, we also detected the levels of VP in plasma. Interestingly, the levels of VP were increased after in vivo electroporation, suggesting that the constructs may be partially expressed in the SON neurons co-expressing both OT and VP. Moreover, the increase of VP may allow the rats to retain body fluid, so that the body weight was slightly increased by overexpressing Syn Ia in the HNS. In conclusion, the constructs can be specifically expressed into the OT neurons and the SV/MV-specific protein Syn Ia can increase the OT release to peripheral plasma.