Dysbindin在發育過程中調控視網膜波的作用機制

The Mechanism of Dysbindin in Regulating Retinal Waves during Development

鄭子霖(Tzu-Lin Cheng)

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(2020年)

https://doi.org/10.6342/NTU202002550

摘要

思覺失調症是一種伴隨許多嚴重症狀的神經發育疾病，包括視覺迴路的異常；已知Dysbindin為一重要的思覺失調症易感蛋白，但Dysbindin在視覺迴路的發育中所扮演的角色迄今未明。視覺迴路的發育需要視網膜波 (一種模式化的神經自發性放電現象)；以出生後一週的大鼠視網膜為例，星狀無軸突細胞 (SACs)會自發性產生神經衝動，分泌乙醯膽鹼和GABA並至周圍其他SACs和視網膜神經節細胞 (RGCs)，產生第二期視網膜波。研究已經證實，第二期視網膜波在調控視覺迴路的發育當中扮演著重要的角色。然而，目前仍為未知Dysbindin是否參與在調控第二期視網膜波的機制當中。實驗室先前利用不具細胞專一性的分子擾動技術，發現大鼠視網膜中Dysbindin的表現量會調控第二期視網膜波的時空性質，但作用機制則需更深入的研究；因此，本研究深入探討Dysbindin調控第二期視網膜波的細胞與分子機制。首先，我們發現在大鼠視網膜中，Dysbindin的蛋白表現量在第二期視網膜波時期較高。利用免疫螢光染色，發現Dysbindin主要分布於視網膜的內部叢狀層和神經節細胞層，並表現於SACs和RGCs中。我們進一步利用SAC和RGC的專一引子，在SACs和RGCs中分別過量或消耗表現Dysbindin，再用鈣離子實像錄影並分析第二期視網膜波的時空性質。結果發現，Dysbindin在SACs中的表現量並不會影響第二期視網膜波的時空性質；然而，在RGCs中，過量表現Dysbindin降低了第二期視網膜波的頻率、波的大小、和同步性；而消耗Dysbindin的表現量則僅降低第二期視網膜波的大小。為了深入研究Dysbindin調控第二期視網膜波的分子機制，我們進一步分析視網膜中與胞吐作用相關的蛋白表現量是否會受到分子擾動Dysbindin的影響。結果發現在視網膜的層級中無法看出Dysbindin和其他蛋白表現量的變化；但在單一細胞的層級中，我們發現了Dysbindin在SACs或RGCs中過量或消耗表現的證據。此外，在RGCs中，隨著Dysbindin的表現量增加或減少，SNAP-25的表現量會同步變化。另外，我們也發現在RGCs中過量表現Dysbindin，會降低SACs中Dysbindin和 SNAP-25的表現量，表示RGCs中的Dysbindin可能存在著逆向調控SACs的作用機制。最後，我們利用Mirror Tree蛋白交互作用預測法輔以免疫共沉澱實驗，發現在發育的大鼠視網膜中，Dysbindin會和SNAP-25有交互作用；表示Dysbindin可能是透過與SNAP-25的交互作用，來調控第二期視網膜波。綜合以上結果，我們發現Dysbindin主要透過RGCs調控第二期視網膜波，並且會調控視網膜細胞中SNAP-25的表現量。因此，本研究發現Dysbindin參與神經迴路發育的作用機制，為未來研究思覺失調症的致病分子機制，提供一個新的方向。

關鍵字

神經發育 ； 思覺失調症 ； Dysbindin ； 第二期視網膜波 ； SNAP-25

並列摘要

Schizophrenia (SCZ) is a neurodevelopmental disease with several symptoms including visual circuit defects. One of the major SCZ susceptibility proteins is Dystrobrevin-binding protein (Dysbindin), but how Dysbindin involves in the development of visual circuits remains unknown. During the first postnatal week, developing rat retinas display the patterned spontaneous activity, termed stage II retinal waves, which are initiated by starburst amacrine cells (SACs) spontaneously releasing acetylcholine (ACh) and -amino butyric acid (GABA) to neighboring SACs and retinal ganglion cells (RGCs), further propagating to central brain. Stage II retinal waves play a critical role in establishing the precise visual circuits. However, the role of Dysbindin in regulating stage II retinal waves remains unclear. By using non-cell-type-specific molecular perturbation, we found that changing the Dysbindin levels in the developing rat retina regulates the spatiotemporal properties of stage II retinal waves. However, the underlying mechanisms are not clear. Here, we aimed to identify the cellular and molecular mechanisms for Dysbindin to regulate stage II retinal waves. First, we found that the expression levels of Dysbindin in rat retinas were relatively high during the stage II wave period compared to adulthood. With immunofluorescence, we found that Dysbindin was localized to the inner plexiform layer (IPL) and ganglion cell layer (GCL), particularly in SACs and RGCs. To identify whether Dysbindin may regulate stage II retinal waves in SACs or RGCs, we combined the cell-type-specific molecular perturbation (the Brn3b promoter for RGCs and the mGluR2 promoter for SACs, respectively) and live Ca2+ imaging to analyze the wave spatiotemporal properties. We showed that overexpressing Dysbindin in RGCs, but not in SACs, dramatically down-regulates the wave spatiotemporal properties, suggesting that Dysbindin regulates stage II retinal waves via RGCs. Furthermore, using immunofluorescence on dissociated cells, we found that the protein levels of SNAP-25 were proportionally changed with the Dysbindin levels in RGCs. Moreover, overexpressing Dysbindin in RGCs decreased the levels of Dysbindin and SNAP-25 in SACs, suggesting a retrograde signal may counter balance the presynaptic levels of Dysbindin and SNAP-25 through the RGC-SAC synapses. Finally, using the Mirror Tree prediction and immunoprecipitation, we found that Dysbindin may interact with SNAP-25, previously shown to regulate the properties of stage II retinal waves, suggesting that Dysbindin may regulate stage II retinal waves via modulating the SNAP-25 level and/or via interacting with SNAP-25. Therefore, our results revealed a role of Dysbindin in regulating retinal waves, providing a new direction for studying visual circuit defects in SCZ.

並列關鍵字

Neurodevelopment ； Schizophrenia ； Dysbindin ； Stage II retinal waves ； SNAP-25

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