- 學位論文
探索RBFOX3 (NeuN)在小鼠海馬迴神經迴路中所扮演的角色
Investigating the Roles of RBFOX3 (NeuN) in the Mouse Hippocampal Circuitry
摘要
二十年以來神經元特異性核抗原(Neuronal nuclei, NeuN)被廣泛地使用於神經元的標定。直到2009年，Kim等人確定它是RBFOX3，為具RNA結合能力的FOX蛋白質家族的其中一員。FOX蛋白質家族是一群調控前信使RNA(pre-mRNA)選擇性剪切的蛋白質。近幾年來已經有數個研究指出FOX蛋白質家族的RBFOX1和RBFOX2在腦中所扮演的角色，而過去人類疾病的臨床研究也發現RBFOX3的突變和癲癇、認知障礙、語言及發展遲緩、長睡眠遲滯期和一些自閉症症狀相關。雖然NeuN已經被確認是RBFOX3，它在腦中的生理功能還未能清楚，因此我們使用Rbfox3基因剔除小鼠來研究它在腦中所扮演的角色。我們實驗室過去發現全身性Rbfox3基因剔除小鼠比起野生型小鼠容易引發癲癇症狀，以及有異常的突觸神經傳遞。Rbfox3基因剔除小鼠的海馬迴齒狀回顆粒細胞的微小興奮性和抑制性突觸後電流(mEPSC和mIPSC)表現出頻率的增加，而其電流大小不變。神經突觸傳遞對於神經訊號的傳遞是相當重要的，因此我試著深入研究RBFOX3在神經迴路中所扮演的角色並想找出過去發現Rbfox3基因剔除缺陷的細胞和分子機制。我發現Rbfox3基因剔除小鼠齒狀回顆粒細胞的樹突棘、興奮性神經突觸數量增加，以及樹突長度增加、型態更加複雜。此外，藉由在特定類型神經細胞的Rbfox3基因剔除小鼠，可以讓我們進行Rbfox3基因的遺傳剖析。在抑制性神經細胞特異性地剔除Rbfox3基因的小鼠會有嚴重的自發性癲癇症狀及未成熟時的死亡現象。抑制性神經細胞特異性地Rbfox3基因剔除小鼠的齒狀回顆粒細胞之微小抑制性突觸後電流表現出頻率的增加，而其電流大小不變。興奮性神經細胞和齒狀回顆粒細胞特異性地Rbfox3基因剔除小鼠的齒狀回顆粒細胞之微小抑制性突觸後電流也表現出頻率些微的增加。這些結果說明了RBFOX3在齒狀回迴路的突觸前和突觸後細胞均在正常神經突觸傳遞功能上扮演著一定的角色。另外在生理情況下，抑制性神經細胞特異性地失去Rbfox3基因會造成神經迴路的興奮性過高。這個研究可以幫助我們更加了解RBFOX3在海馬迴神經迴路、Rbfox3基因缺失造成的癲癇症狀之產生所扮演的角色，以及RNA選擇性剪切調控蛋白(如RBFOX3)如何在腦中進行功能。
並列摘要
Neuronal nuclei (NeuN), a neuronal nuclear antigen, is widely used to label neurons for over 20 years. In 2009, Kim et al. identified that NeuN corresponds to a protein known as RBFOX3 (RNA binding protein fox-1 homolog 3), one of the FOX family proteins. FOX proteins are a family of regulators that control the pre-mRNA alternative splicing. In recent studies, the roles in the brain of other FOX family members, RBFOX1 and RBFOX2, have been investigated. Previous research also found that RBFOX3 mutations are linked to human epilepsy, cognitive impairment, speech disorder, developmental delay, long sleep latency and some autistic features. Although NeuN has been identified as RBFOX3, it is still unclear what physiological roles it plays in the brain. Therefore, we use Rbfox3 knockout mice model to investigate the roles of RBFOX3 in the brain. The previous findings in our lab indicated that conventional Rbfox3-/- mice had increased seizure susceptibility and abnormal synaptic transmission. The dentate gyrus granule cells (DGGCs) of Rbfox3-/- mice displayed both increased frequency, but not amplitude, of excitatory and inhibitory postsynaptic current (mEPSC and mIPSC). Synaptic transmission is very important to neuronal signal transduction. For this reason, I attempt to further study the roles of RBFOX3 in neuronal circuitry and to find out the cellular and molecular mechanisms underline previous findings. I found that the DGGCs of Rbfox3-/- mice exhibited increased spine density, excitatory synapse number, and dendritic complexity. Furthermore, with the different kinds of cell type-specific conditional Rbfox3 knockout mice, this can allow us to enable genetic dissection of Rbfox3. Specific deletion of Rbfox3 in inhibitory interneurons caused spontaneous seizure and premature death. The DGGCs of Gad2-Cre::Rbfox3loxP/loxP conditional KO mice displayed increased frequency, but normal amplitude, of mIPSC. Specific deletion of RBFOX3 in excitatory neurons and specific deletion of RBFOX3 in the DGGCs also displayed slight increased mIPSC frequency in DGGCs. These results indicated that the RBFOX3 in presynaptic and postsynaptic neurons of the dentate gyrus circuitry play a role in normal function of synaptic transmission. Moreover, in the physiological condition, GABAergic neuron- specific loss of Rbfox3 cause circuitry hyper-excitability. This study can help us more understand the roles of RBFOX3 in hippocampal circuitry, seizure pathogenesis of Rbfox3 defect, as well as how the splicing regulators such as RBFOX3 operate in the brain.