摘要:Dysbindin由思覺失調症易感基因所編碼,涉及思覺失調症的發病。Dysbindin可調節神經元功能,包括胞吐動態、受體運輸、溶酶體相關胞器和樹突棘的形成。然而,Dysbindin如何影響神經網路發育仍然未知。在發育關鍵時期,神經網路會顯示出獨特的“模式化自發性放電現象”,在視覺系統中稱為“視網膜波”。視網膜波出現於視覺開始前的關鍵期(即大鼠出生後一周),此時的視網膜中多為神經母細胞 (neuroblasts),僅含有兩種已分化完全的神經元‒星狀無軸突細胞 (starburst amacrine cells, SACs) 和視網膜神經節細胞 (retinal ganglion cells, RGCs)。視網膜波由SACs釋放傳導物質至RGCs所引發,並以波動狀的時空特性傳至整個視覺網路。視網膜波獨特的時空模式可誘導基因的表現(主要為先天免疫蛋白),使突觸被修飾, RGCs軸突投射到腦中正確位置。擾亂視網膜波被認為是思覺失調症的病因之一,但其中的關鍵基因尚待釐清。我們假設Dysbindin可能為其中主要的關鍵基因:第一,我們之前的研究顯示,大鼠發育中視網膜SACs的25kD突觸體相關蛋白(SN25b)可藉由PKA磷酸化來調節視網膜波的時空模式,導致視覺網路的缺陷;他人文獻指出Dysbindin可與SN25相互作用。第二,我們初步結果顯示,Dysbindin可在發育中視網膜的SACs-RGCs的突觸間表現,改變Dysbindin在發育中視網膜的表現量可調控視網膜波的時空模式。在本研究中,我們盼能解開Dysbindin調控視網膜波模式和視覺網路發育的機制。藉由結合分生技術、視網膜培養、基因轉染、免疫螢光與生化、細胞電生理、細胞影像、和軸突追踪,我們將回答以下問題。Dysbindin調節視網膜波模式的細胞機制為何?是影響SACs的釋放機制?或影響RGCs的反應機制?能否調控細胞中溶酶體或外泌體的輸出? 第二, Dysbindin調節視網膜波模式的分子機制為何?是透過調控先天免疫蛋白?與SACs中SN25b交互作用?還是有其他蛋白與Dysbindin交互作用?最後,Dysbindin可否調控RGCs到腦中的正確投射?藉回答這些問題,我們可了解Dysbindin調控視覺網路發育的機制,釐清思覺失調症的發病機理,並理解神經網路發育的通則。
Abstract: Dysbindin, encoded by a schizophrenia susceptibility gene, involves in schizophrenia pathogenesis. Previous studies revealed that Dysbindin regulates neuronal function, including exocytotic kinetics, receptor trafficking, biogenesis of lysosome-related organelles, and dendritic spine formation. However, how Dysbindin affects neural circuit development remains completely unknown. During the developmental critical period, all developing neural circuits display a unique “patterned spontaneous activity”, such as “retinal waves” in the developing visual system. Retinal waves occur within a developmental critical period prior to visual experience, i.e., the first postnatal week in rodents. At this stage, retinas consist of mainly neuroblasts and only two types of differentiated neurons, i.e., starburst amacrine cells (SACs) and retinal ganglion cells (RGCs). Retinal waves are initiated by neurotransmitter release from presynaptic SACs onto postsynaptic RGCs, propagating through the entire visual system with distinct spatiotemporal properties, inducing activity-dependent gene expression (mainly innate immune proteins), finally resulting in synaptic refinement and precise RGCs’ axonal projection to brain targets. Perturbations of retinal waves are thought to be associated with schizophrenia etiology, but the key genes remain to be identified. Here, we hypothesize that Dysbindin could be the major key gene, based on the following reasons: First, our previous studies showed that synaptosome-associated protein of 25 kD (SNAP-25b or SN25b) in developing rat SACs can regulate the spatiotemporal patterns of retinal waves via PKA-mediated phosphorylation, leading to defects in visual circuit development. Moreover, Dysbindin is found to interact with SN25b. Second, our preliminary results show that Dysbindin localizes to the synapses of SACs-RGCs in the developing retina. Manipulating the expression levels of Dysbindin in the developing retina can regulate the spatiotemporal pattern of retinal waves. In this study, we hope to elucidate how Dysbindin modulates retinal wave patterns and visual circuit refinement. By combining molecular biology, primary retinal explant culture, ex vivo/in vivo transfection, immunofluorescence and biochemical experiments, whole-cell patch clamp, live imaging, and axonal tracing, we will answer the following questions. First, what is the cellular mechanism by which Dysbindin regulates retinal wave patterns? By affecting the release of presynaptic SACs? By affecting the responsiveness of postsynaptic RGCs? Or by regulating the export of lysosomes/exosomes from SACs/RGCs? Second, what is the molecular mechanism by which Dysbindin regulates retinal wave patterns? By regulating the signaling of innate immune proteins? By interacting with SN25b in SACs? Or are there other proteins that interact with Dysbindin to regulate the pattern of retinal waves? Finally, can Dysbindin regulate the RGCs’ axonal projection into central brain targets? Through answering these questions, we will understand how Dysbindin regulates the patterns of retinal waves and the development of visual circuits. The results from this grant proposal will clarify the pathogenesis of schizophrenia and provide the foundation to further understand the general rules for neural circuit development.