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標題: | 半胱胺酸串鍊蛋白的磷酸化對於視網膜波的影響 The Effects of Phosphorylation of Cysteine String Protein on Retinal Waves |
作者: | Ching-Feng Chen 陳境峰 |
指導教授: | 王致恬(Chih-Tien Wang) |
關鍵字: | 半胱胺酸串鍊蛋白,視網膜波,星狀無軸突細胞,視網膜節細胞,鈣離子顯像技術, cysteine string protein,retinal waves,starburst amacrine cells,retinal ganglion cells,calcium imaging, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | 視覺系統在發育的關鍵時期會產生一種自發性的放電現象,稱為「視網膜波」,對於神經迴路的正確連結極為重要。視網膜波的形成是透過突觸前神經元—星狀無軸突細胞—釋放乙醯膽鹼,經由突觸後神經元—視網膜節細胞—接收後而產生。先前的研究顯示,發育中的星狀無軸突細胞能週期性地產生神經衝動並進行鈣離子調控的胞吐作用,釋放興奮性神經傳導物質例如乙醯膽鹼和γ-丁氨基酪酸,進而引發視網膜波。然而,其中仍不甚了解的是,星狀無軸突細胞中鈣離子調控之胞吐作用的改變如何能影響視網膜波的時空特性。
半胱胺酸串鍊蛋白被發現能幫助SNARE蛋白進行正確摺疊,SNARE蛋白已被證實為胞吐作用中膜融合的參與分子,因此半胱胺酸串鍊蛋白可藉由影響SNARE蛋白來調控神經傳導物質的釋放。此外,半胱胺酸串鍊蛋白能被蛋白質激酶A所磷酸化,而這種蛋白質激酶在視網膜波發生的時期會被高度活化。這些結果暗示,星狀無軸突細胞或許能經由細胞內訊息傳遞路徑以調控半胱胺酸串鍊蛋白的磷酸化及其功能,進而影響視網膜波的時空特性。 在此論文中,我們研究半胱胺酸串鍊蛋白的磷酸化是否能影響視網膜波的時空特性。首先,我們使用免疫螢光染色證明半胱胺酸串鍊蛋白能在新生老鼠的內網狀層中表現,在分離的視網膜細胞中,更發現半胱胺酸串鍊蛋白能表現在星狀無軸突細胞內,說明了半胱胺酸串鍊蛋白存在於星狀無軸突細胞中,可能可以調控其神經傳導物質釋放的機制。此外,定量聚合酶鏈鎖反應的實驗結果顯示,發育中的視網膜內最主要的半胱胺酸串鍊蛋白為α型。為了更進一步研究突觸前星狀無軸突細胞中半胱胺酸串鍊蛋白的磷酸化是否會影響視網膜波,我們將特定基因(半胱胺酸串鍊蛋白或其磷酸化突變株)利用專一性啟動子大量地表現於星狀無軸突細胞之中,再利用鈣離子顯像技術記錄視網膜節細胞層的鈣離子變化以偵測視網膜波的時空特性。我們發現當星狀無軸突細胞表現半胱胺酸串鍊蛋白磷酸化缺陷型S10A時,會顯著降低視網膜波的發生頻率;其餘控制組、半胱胺酸串鍊蛋白野生型及磷酸化模擬型S10D & S10E則否。相較之下,半胱胺酸串鍊蛋白對於視網膜的空間性質只有較小的影響。全細胞膜電壓固定實驗顯示,半胱胺酸串鍊蛋白磷酸化缺陷型S10A降低視網膜節細胞內突觸後電流頻率和其斜率,但不影響視網膜節細胞的電生理特質,顯示大量表現磷酸化缺陷型S10A所造成的結果是來自突觸前星狀無軸突細胞中神經傳導物質釋放的改變所致。有鑑於半胱胺酸串鍊蛋白磷酸化缺陷型S10A會降低視網膜波的頻率而不影響其空間性質,我們的研究顯示半胱胺酸串鍊蛋白的磷酸化,在調控視網膜波的時間性質上,扮演著重要角色。 During a developmental critical period, the visual system displays a robust spontaneous activity termed “retinal waves”, essential for neural circuit refinement. These waves are initiated by releasing neurotransmitters from presynaptic starburst amacrine cells (SACs) onto postsynaptic retinal ganglion cells (RGCs). Previous studies showed that the developing SACs can periodically fire action potentials and undergo Ca2+-regulated exocytosis, thus releasing excitatory neurotransmitters such as acetylcholine (ACh) and γ-amino butyric acid (GABA) and inducing periodic retinal waves. However, little is known regarding how altering the Ca2+-regulated exocytosis in SACs affects the spatial or temporal properties of retinal waves. Cysteine string protein (CSP) was found to ensure the correct folding of fusion machinery [i.e., soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins], thus playing an important role in regulating neurotransmitter release. In addition, CSP can be phosphorylated by protein kinase A (PKA) that is highly activated during retinal waves. These results suggest that the intracellular signaling in SACs may modulate the function of CSP and thus regulate neurotransmitter release during retinal waves. In this study, we investigated how phosphorylation of CSP affects the spatial or temporal properties of retinal waves. First, we used immunofluorescence staining to show that CSP was expressed in the inner plexiform layer (IPL) of the neonatal rat retina. Further immunostaining of dissociated retinal cells confirmed that the expression of CSP was localized to presynaptic SACs, implying that CSP may involve in regulating neurotransmitter release from SACs. The quantitative polymerase chain reaction (qPCR) experiment showed that CSPα was the dominant isoform in the developing rat retina. To investigate whether phosphorylation of CSPα in presynaptic SACs affects retinal waves, we targeted gene expression to SACs by the metabotropic glutamate receptor type II promoter (pmGluR2). After overexpression of CSP or its phosphomutants in SACs, subsequent Ca2+ imaging was performed in the RGC layer to detect the spatial and temporal properties of wave-associated spontaneous Ca2+ transients. We found that the frequency of Ca2+ transients was significantly decreased by the phosphodeficient mutant (CSPα-S10A), but not by the wild-type CSPα (CSPα-WT) or the phosphomimetic mutants (CSPα-S10D and CSPα-S10E) compared to the control. In contrast, the CSPα phosphodeficient mutant had a relatively minor effect on the spatial correlation of spontaneous Ca2+ transients over distance. Whole-cell voltage-clamp recordings demonstrated that the CSPα phosphodeficient mutant reduced the frequency and the slope of wave-associated postsynaptic currents (PSCs), but not altered the electrical properties of postsynaptic RGCs, suggesting that the effects by overexpressing the CSPα phosphodeficient mutant were due to the change in the release from presynaptic SACs. Given that the CSPα phosphodeficient mutant down-regulates the frequency but does not significantly alter the spatial correlation of retinal waves, our results suggest that phosphorylation of CSPα may play an important role in regulating the temporal properties of retinal waves. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/57522 |
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顯示於系所單位: | 分子與細胞生物學研究所 |
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