在成年雄鼠下視丘-腦下垂體系統中研究Synapsin Ia 調控神經胜肽的釋放所扮演的角色

The Role of Synapsin Ia in Regulating Neuropeptide Release from the Hypothalamic-Neurohypophysial System of Adult Male Rats

張詠淇(Yung-Chi Chang)

指導教授 ： 王致恬

國立臺灣大學/生命科學院/分子與細胞生物學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.02393

摘要

下視丘-腦下垂體系統由magnocellular neurons (MCNs)所組成，製造神經傳導物質(催產素和血管加壓素)，分別沿著神經元的樹突釋放到腦脊髓液中或是沿著軸突釋放都周邊血液系統中。在下視丘-腦下垂體系統中有兩種囊泡包裹神經傳導物質，分別是large-dense core vesicles (LDCVs)和microvesicles (MVs)。先前研究指出一種弱鈣離子感應蛋白-Synaptotagmin IV (Syt IV)表現在LDCVs和MVs中，並且參與鈣離子調控性胞吐作用。然而在下視丘-腦下垂體系統中，MVs不論在大小、型態和分子組成上與其他神經組織中的synaptic vesicles (SVs)相類似，但MVs其包裹的神經傳導物質和在下視丘-腦下垂體系統中的功能尚未了解。因此，我們假設在成年雄鼠的下視丘-腦下垂體系統中，一種SV/MV的蛋白-Synapsin Ia (Syn Ia)可能會影響神經胜肽從LDCVs中釋放。首先，我們利用免疫螢光染色，可以觀察到Syn Ia在視上核和腦下腺後葉的表現不會和包覆在LDCVs中的催產素和血管加壓素重疊，證明Syn Ia 是在MVs中表現。接著，我們利用活體電穿孔方式，將不同種的DNA轉染到視上核中。轉染的DNA種類包含Syt I、 Syt IV、wild-type Syn Ia和仿效無法被磷酸化的Syn Ia (Syn Ia-S62A). 我們利用酵素連結免疫吸附試驗，來偵測催產素和血管加壓素在腦脊髓液和周邊血液中的濃度，因為轉染不同種DNA而造成的影響。研究結果顯示，Syn Ia 促進催產素在腦脊髓液和周邊血液中的含量增加，並且這個情形會因為Syn Ia-S62A而反轉。當共同轉染Syt IV和Syn Ia時，仍然會增加催產素在腦脊髓液和周邊血液中的含量，並且一樣會被共同轉染Syt IV 和Syn Ia-S62A反轉。因此，證明Syn Ia是藉由第62個胺基酸的位置磷酸化與否來調控催產素在腦脊髓液和周邊血液中的釋放。另一方面，血管加壓素釋放到腦脊髓液和周邊血液中，也是藉由Syn Ia第62個胺基酸的位置磷酸化來調控。此外，鈣離子感應蛋白-Syt I會增加血管加壓素在周邊血液的釋放量，並且會被Syt IV所反轉。綜合上述，可以得知在下視丘-腦下垂體系統中，Syn Ia調控催產素和血管加壓素的釋放是藉由第62個胺基酸的位置磷酸化與否，並且仿效無法被磷酸化的Syn Ia會抑制MV/SV的傳遞，來調控LDCVs的釋放。

關鍵字

synapsin ； 下視丘-腦下垂體系統 ； 催產素 ； 血管加壓素 ； large-dense croe vesicle ； synaptic vesicle ； 活體電穿孔

並列摘要

The magnocellular neurons (MCNs) of hypothalamic neurohypophysial system (HNS) release neuropeptides (oxytocin, OT; vasopressin, VP) into the cerebrospinal fluid (CSF) and peripheral plasma from their somatodendrites and axonal terminals, respectively. The vesicles in the HNS fall into two distinct classes, neuropeptide-laden large-dense core vesicles (LDCVs) and microvesicles (MVs). A previous study suggested that a poor Ca2+ sensor protein, synaptotagmin IV (Syt IV), localizes to both classes of vesicles and alters Ca2+-regulated exocytosis of both vesicles. Although MVs are similar to synaptic vesicles (SVs) in size, morphology and molecular composition, their contents and functions in the HNS remain unknown. Here, we examined whether a SV/MV protein, synapsin Ia (Syn Ia), may participate in regulation of neuropeptide release from LDCVs in the HNS of adult male Sprague-Dawley rats. We found that Syn Ia did not colocalize with either oxytocin (OT) or vasopressin (VP)-containing LDCVs in the MCNs or pituitary nerve terminals, suggesting that Syn Ia localizes to SVs/MVs exclusively. To determine the role of SVs/Syn Ia in HNS, we conducted molecular perturbation by in vivo electroporation to unilaterally transfect supraoptic nucleus with various DNA plasmids, including Syt I, Syt IV, wild-type Syn Ia, and its phospho-deficient mutant (Syn Ia-S62A) to abolish SV/MV recruitment to plasma membrane. Sham control was the vector carrying the transfection marker EGFP. To detect the OT/VP changes following various molecular perturbations, CSF and plasma were collected for ELISA measurements before and after in vivo electroporation. Syn Ia significantly increased the OT levels in CSF/plasma, while Syn Ia-S62A did not alter the OT levels in CSF/plasma. Furthermore, cotransfecting Syt IV and Syn Ia also increased the OT levels in CSF/plasma, while this effect was abolished by cotransfecting Syt IV and Syn Ia-S62A. These results suggest that the defective S62 phosphorylation may abolish the Syn Ia-enhanced OT central/peripheral release. Transfection of Syn Ia alone, or Syn Ia together with Syt IV also increased the VP levels in CSF, and this Syn Ia-enhanced VP central release was abolished by defective S62 phosphorylation. In addition, Syn Ia significantly increased the VP levels in plasma, while Syn Ia-S62A slightly decreased the VP levels in plasma. Moreover, the functional Ca2+ sensor protein, Syt I, significantly increased the VP levels in plasma, while Syt IV (harboring a natural mutation in its C2A Ca2+ binding site) did not alter the VP levels in plasma. However, cotransfecting Syt IV and Syn Ia still increased the VP levels in plasma, while this effect was abolished by cotransfecting Syt IV and Syn Ia-S62A. Thus, even though cotransfection with the poor Ca2+ sensor protein Syt IV, Syn Ia was sufficient to enhance VP peripheral release. Taken together, the defective S62 phosphorylation may abolish the Syn Ia-enhanced neuropeptide central/peripheral release. Given that this Syn Ia phosphodeficient mutant blocks SV/MV recruitment, our results suggest that SV/MV recruitment to plasma membrane may facilitate the trafficking and fusion of the other class of vesicles, LDCVs, in the HNS.

並列關鍵字

synapsin ； hypothalamic-neurohypophysial system ； oxytocin ； vasopressin ； large-dense core vesicles ； synaptic vesicles ； in vivo electroporation

參考文獻

Aihara H, Miyazaki J (1998) Gene transfer into muscle by electroporation in vivo. Nature biotechnology 16:867-870.

Akaneya Y, Jiang B, Tsumoto T (2005) RNAi-induced gene silencing by local electroporation in targeting brain region. Journal of neurophysiology 93:594-602.

Ashford JW, Soultanian NS, Zhang SX, Geddes JW (1998) Neuropil threads are collinear with MAP2 immunostaining in neuronal dendrites of Alzheimer brain. Journal of neuropathology and experimental neurology 57:972-978.

Bergquist F, Ludwig M (2008) Dendritic transmitter release: a comparison of two model systems. Journal of neuroendocrinology 20:677-686.

Blume A, Bosch OJ, Miklos S, Torner L, Wales L, Waldherr M, Neumann ID (2008) Oxytocin reduces anxiety via ERK1/2 activation: local effect within the rat hypothalamic paraventricular nucleus. The European journal of neuroscience 27:1947-1956.