探索RBFOX3 (NeuN)在小鼠大腦中所扮演的角色

Abstract

RBFOX3 (NeuN)是一個Fox家族的成員之一，能調控選擇性剪接的事件，它只表現在神經系統裡。在先前研究發現，病患若是此基因有所突變，會有癲癇或是認知上的問題。此外，也發現它能夠調控Numb的選擇性剪接，進而影響神經發育。雖然Fox家族已經被發現很久，RBFOX1與RBFOX2的功能也逐漸解謎，但是目前並未有研究能夠解釋RBFOX3在腦中所扮演的角色是甚麼。 在本研究中，利用基因轉殖的技術將Rbfox3剔除所得到的小鼠，探索Rbfox3在海馬迴所扮演的角色。首先，我們發現此小鼠的大腦重量較野生老鼠輕，但是兩者間的體重是沒有差異的。因為齒狀回是能夠過濾來自海馬迴裡過於興奮的訊號以及海馬迴同步的現象，且此區域也是調控學習與記憶和認知程序的地方，所以選擇看齒狀回被影響的情況。結果顯示Rbfox3剔除鼠在此處其神經完整性是較差的。另外，在行為實驗中也顯示此種老鼠相對於野生種來說較不焦慮。透過卡英酸(Kainic acid)所引發的癲癇實驗所得到的結果顯示與之前研究相同的結果：Rbfox3剔除鼠較容易引發癲癇，引發的結果也較為嚴重。此外，我們同時量測此老鼠的神經細胞電生理，發現他的興奮性與抑制性的神經傳導頻率變高，但是內生性刺激的性質與對照組是沒有差異的。最後，我們利用RNA-Seq的技術來搜索Rbfox3可能的下游分子，發現在海馬迴中Arc, Egr4, Per1表現量都隨著Rbfox3的消失而上升。 本研究為第一個在活體內探索Rbfox3在腦中所扮演的角色與功能，能為臨床上所發現的病症帶來一些可能的致病機制。

RBFOX3, wellknown as NeuN, is one of the Fox family proteins, which is a splicing regulator. Its disruption has been identified in patients with epilepsy and cognitive impairment. RBFOX3 promotes neuronal differentiation through alternative splicing of Numb pre-mRNA during brain development. In recent study, the roles of RBFOX1 and RBFOX2 have been more understanding for its function of splicing; however, the researches about the role of RBFOX3 are still large unknown. To address this critical question, we decided to further explore RBFOX3 in the hippocampus of mice. We first validated and characterized Rbfox3 knockout mice. Rbfox3-/- mice showed similar body weight, however, they exhibited decreased brain weight in comparison to their wild-type counterpart, regardless of gender. The dentate gyrus (DG) of the hippocampus serves as a filter for protecting against hyperexcitability and hippocampal synchronization leading to temporal lobe seizures. In addition, the DG plays an important role in learning, memory and cognition, in addition to modulating sensorimotor gating, which is involved in cognitive processes. The hippocampal DG of Rbfox3-/- mice exhibited a deficit in neuronal integrity. Rbfox3-/- mice also exhibited deficits in anxious behavior during elevated T-maze and novelty suppressed feeding behavioral tests, increased seizure susceptibility and deficits in spatial reversal learning. Therefore, Rbfox3-/- mice could replicate the symptoms of patients with disrupted RBFOX3. Furthermore, Rbfox3-/- mice exhibited increased frequency, but not amplitude, of excitatory and inhibitory neurotransmission. Neuronal intrinsic excitability is intact in Rbfox3-/- mice. We also identified three genes whose expression was increased in the hippocampus of Rbfox3-/- mice: Egr4, Arc and Per1. Interestingly, we also found that three genes were upregulated in the striatum of Rbfox3-/- mice: Ppp1r1b, Adora2a, Drd2. This study can help us to know more detail information in vivo evidence of the physiological roles of RBFOX3 in neuronal circuitry in the DG of the hippocampus.