在星狀無軸突細胞中的半胱胺酸串鍊蛋白磷酸化對發育中大鼠視網膜的基因表現的影響

Abstract

發育中的神經系統在有感覺經驗之前，模式化自發性作用又稱做視網膜波對於建立功能性的神經網路很重要。第二期視網膜波發生於囓齒類動物出生後一周間，被發現可以促使一些先天性免疫蛋白的表現，其中包含了第一型主要組織相容性複合體 (major histocompatibility complex class I; MHCI)，MHCI與突觸的修剪以及視神經節細胞 (RGCs) 軸突投射到腦區所導致的雙眼專一性投射分層，造成雙眼視野的隔離。這時期的視網膜波是由突觸前的星狀無軸突細胞 (SACs) 釋放神經傳導物質到鄰近的SACs或是RGCs，造成像波一樣的傳遞，因此有獨特的空間及時間傳播特性。伴隨著視網膜波的產生，視網膜神經節細胞層上會有大量的鈣離子流入。目前還不知道這些受到視網膜波調控的免疫基因表現主要是透過視網膜的空間還是時間特性。在先前的研究中發現半胱胺酸串鍊蛋白(CSP) 在突觸前神經元細胞內的磷酸化修飾可以增加神經傳導物質的釋放以及視網膜波的頻率，並且不會影響到視網膜波的空間傳播特性。因此，透過改變突觸前神經元內的CSP磷酸化程度可以用來研究視網膜波時間特性對於這些免疫基因表現的影響。在本篇研究中，我們探討了發育中大鼠視網膜內，視網膜波的時間特性如何調控活性依賴型的基因(activity-dependent gene)表現。我們將CSP 專一性的表現到SAC中，並且發現當CSP過量表現於SAC時相對於控制組會使得這些先天性免疫蛋白的表現增加了，但是若將無法被磷酸化的CSP突變株過量表現到SAC就無法增加這些先天性免疫基因的表現，除此之外，我們利用追蹤MHCI上游轉錄因子，磷酸化CREB (pCREB)的表現量，發現CSP-WT表現在SAC中會使突觸後神經元的pCREB增加，而突變株與對照組沒有顯著差異，因此，我們得知在SAC內，CSP的磷酸化可以增加視網膜波的頻率並且調控先天性免疫基因在視網膜中的表現。這些結果代表視網膜波的時間特性對於發育中大鼠視網膜內的活性依賴型基因表現扮演重要的角色。

Prior to sensory experience, patterned spontaneous activity (termed retinal waves in the developing visual system) is essential for establishing functional neural circuits. In rodents, retinal waves during the first postnatal week up-regulate the expression of innate immune proteins, such as histocompatibility complex class I (MHCI), leading to synaptic refinement and eye-specific segregation of retinogeniculate projection. These waves are initiated by presynaptic starburst amacrine cells (SACs) releasing neurotransmitters to neighboring SACs or retinal ganglion cells (RGCs), displaying unique spatial and temporal patterns, with wave-like propagation of correlated firings and Ca2+ transients in the RGC layer. However, which pattern (spatial or temporal) of retinal waves is important for this activity-dependent gene expression remains unknown. We previously found that phosphorylation of cysteine string protein-α (CSPα) in presynaptic cells increases neurotransmitter release and wave frequency, without altering the spatial correlation of retinal waves. Thus, the effects by altering the CSPα phosphorylated state in wave-initiating cells can reflect how the wave temporal pattern regulates activity-dependent gene expression. In this study, we determine how the temporal pattern of retinal waves regulates activity-dependent gene expression in the developing rat retina. By utilizing the cell type-specific molecular perturbation, we found that overexpression of wild-type CSPα in SACs increased the expression of innate immune proteins compared to control, but overexpression of the CSPα phosphodeficient mutant (CSPα-S10A) in SACs did not. In addition, CSP-WT in SACs also increased the expression of the upstream transcription factor, pCREB, in postsynaptic cells compared to control or CSPα-S10A. Therefore, through increasing wave frequency, phosphorylation of CSPα in SACs regulates the retinal expression of innate immune genes. These results suggest that the temporal pattern of retinal waves is important for activity-dependent gene expression in the developing rat retina.